JP7336091B2 - Simple genetic testing method, copy number measurement method, and supporting technology - Google Patents

Description

The present invention relates to genetic analysis methods and kits. More specifically, it relates to a simple gene copy number measuring method and its support technology.

Single nucleotide polymorphisms (SNPs), microsatellite polymorphisms (STRPs), and copy number polymorphisms (CNPs) having base sequences different from those of the wild type exist among individual organisms. These single nucleotide polymorphisms, microsatellite polymorphisms and copy number polymorphisms are known to cause differences in individual basal metabolism, properties, diseases, etc. Since testing leads to the prediction of diseases that may occur in the future, it is also regarded as clinically important. Therefore, various researches are being conducted to test whether or not a specific gene in the DNA of an individual is mutated, and it is widely used as a method for amplifying genes contained in test samples such as biological samples and clinical samples. The PCR method used in this field has also been applied to SNP analysis, microsatellite polymorphism and copy number variation (CNP) analysis.

Efficient treatment by avoiding side effects by examining genetic SNPs, microsatellite polymorphisms, and copy number polymorphisms before administration, and determining the appropriate drug dosage based on the type of the gene. It will be possible to realize medical care that seeks to obtain effects, in other words, more appropriate medical care according to the constitution of each patient, which is called “tailor-made medical care,” “order-made medical care,” or “personalized medical care.” It is also expected to reduce medical expenses by dealing with side effects and reducing inappropriate medications. Thus, there are great expectations for the practical application and spread of diagnosis using SNPs, microsatellite polymorphisms, and differences in copy number polymorphisms.

The present inventors developed a gene detection method using a water-degradable carrier and filed a patent application (Patent Document 2).

JP 2005-245272 A Japanese Patent No. 5875230

The present invention provides a kit for preparing a sample for nucleic acid amplification from a sample containing a nucleic acid-containing biomaterial. By using this kit, it is possible to identify the copy number after nucleic acid amplification, detect the existing copy number (including 0) of a specific gene, and identify copy number variation ( Iafrate AJ, Feuk L, Rivera MN et al. Detection of large-scale variation in the human genome. Nat Genet. 2004. 36: 949-51., Sebat J, Lakshmi B, Troge J et al. Polymorphism in the human genome. Science. 2004. 305: 525-528).

As another method for collecting test samples, a method has been reported in which cells of the oral mucosa are scraped off with a cotton swab or the like. However, in this case, it is necessary to extract DNA from the cells held in the cotton swab and add the extracted DNA solution to a separately prepared amplification reaction solution. Mistakes and contamination may occur, so it is not the best method. Therefore, the present invention provides a sample containing a nucleic acid-containing biomaterial, which contains (A) a carrier capable of binding a nucleic acid-containing biomaterial and (B) a substrate having a lower ability to bind the nucleic acid-containing biomaterial than the carrier. The above problems have been solved by providing a kit for preparing a sample for nucleic acid amplification and developing an application thereof.

Accordingly, the present invention provides:
(Item 1)
(A) a carrier capable of binding a nucleic acid-containing biomaterial;
(B) A kit (sample preparation kit) for preparing a sample for nucleic acid amplification from a sample containing a nucleic acid-containing biomaterial, comprising a base material having a lower binding ability for the nucleic acid-containing biomaterial than the carrier.
(Item 2)
Kit according to item 1, wherein the substrate (B) is configured to provide a sample containing a range of amounts of nucleic acid-containing biomaterial.
(Item 3)
3. The kit of item 2, wherein the surface area of said substrate (B) is sufficient to provide said range of amounts.
(Item 4)
The kit according to any one of items 1 to 3, wherein the carrier (A) allows nucleic acid amplification when placed in water.
(Item 5)
The kit according to any one of items 1 to 4, wherein the carrier (A) allows optical detection.
(Item 6)
The kit according to any one of items 1 to 5, wherein the nucleic acid in the sample after nucleic acid amplification is optically detected.
(Item 7)
The kit according to any one of items 1 to 6, wherein the carrier (A) is a water-decomposable carrier.
(Item 8)
The kit according to any one of items 2 to 7, wherein the amount within a certain range is an amount capable of discriminating the copy number after nucleic acid amplification.
(Item 9)
Items 2 to 8, wherein the fixed range amount is within X times to 1/X of the reference amount, and X ² is the difference in magnification that can be identified when the copy number differs by 1 in the nucleic acid amplification. The kit according to any one of the above.
(Item 10)
The kit according to any one of items 1 to 9, wherein the substrate (B) is a substrate that supports a nucleic acid-containing biomaterial without binding.
(Item 11)
The base materials include polyurethane, polyvinyl alcohol (PVA), polyethylene, chitin, chitosan, collagen, polyvinylidene difluoride (PVDF), polypropylene, cellulose and its derivatives, paper, collagen, gelatin, urethane, silicon and absorbent cotton ( 11. The kit according to any one of items 1 to 10, comprising at least one material selected from the group consisting of cellulose absorbent cotton, acetate absorbent cotton), or combinations thereof, or mixtures thereof.
(Item 12)
12. The kit according to any one of items 2 to 11, wherein said constant range amount is achieved by removing said carrier in a constant area.
(Item 13)
13. The kit according to item 12, wherein the surface area of the substrate (B) is equal to or greater than the fixed area.
(Item 14)
14. Kit according to item 12 or 13, wherein the substrate (B) is of a shape sufficient to cover the fixed area.
(Item 15)
15. The kit according to any one of items 12-14, wherein said constant area is achieved by cutting said constant area with a cutting device capable of cutting said constant area.
(Item 16)
16. The kit of item 15, wherein the resector is selected from a biopsy trepan, and a punch.
(Item 17)
17. The kit of any one of items 2-16, wherein said range amount is achieved by providing said sample in a constant amount.
(Item 18)
18. The kit according to any one of items 1-17, wherein the carrier is water-disintegrable paper.
(Item 19)
The kit according to any one of items 1 to 18, wherein the nucleic acid amplification is a nucleic acid amplification method that causes non-specific amplification.
(Item 20)
A kit for amplifying nucleic acid without purification (nucleic acid amplification kit), comprising the kit according to any one of items 1 to 19 and (C) a reagent for nucleic acid amplification.
(Item 21)
21. The kit according to item 20, wherein the reagents for nucleic acid amplification include primers, nucleic acid amplification enzymes, and necessary buffers.
(Item 22)
22. The kit according to any one of items 1 to 21, wherein (A) further comprises a non-water-absorbing material and a water-absorbing material.
(Item 23)
(X) a non-water absorbent material;
(Y) a carrier capable of binding a nucleic acid-containing biomaterial;
(Z) a device for drying a biological sample, comprising a water absorbent material;
(Item 23A)
(X) a non-water absorbent material;
(Y) a carrier capable of binding a nucleic acid-containing biomaterial;
A device for drying a biological sample, comprising:
(Item 24)
The device according to item 23 or 23A, wherein the carrier (Y) allows nucleic acid amplification when placed in water.
(Item 25)
A device according to item 23, or 23A or 24, wherein said carrier (Y) allows optical detection.
(Item 26)
26. The device of any one of items 23, 23A, or 24-25, wherein the carrier (Y) is a water-disintegratable carrier.
(Item 27)
The kit of item 18 or the kit of items 23, 23A, or 24-26, wherein said non-water absorbent material is arranged to hold said carrier, and said non-water absorbent material is removably coupled to said water absorbent material. A device according to any one of clauses.
(Item 28)
28. The kit or device of any one of items 22, 23, 23A, or 24-27, wherein said non-water absorbent material is connected to said water absorbent material via a removable adhesive member.
(Item 29)
29. The kit or device of any one of items 22, 23, 23A, or 24-28, wherein said non-water absorbent material is arranged to surround said carrier.
(Item 30)
30. The kit or device of any one of items 22, 23, 23A, or 24-29, wherein said carrier is in contact with said non-bibulous material and said bibulous material.
(Item 31)
31. The kit or device of any one of items 22, 23, 23A, or 24-30, wherein the carrier is arranged so as not to be in direct contact with the water-absorbent material.
(Item 32)
A method for detecting a specific gene contained in a nucleic acid-containing biomaterial and/or the copy number of the specific gene, comprising:
A step of providing a test sample containing the nucleic acid-containing biomaterial by using (A) a carrier capable of binding the nucleic acid-containing biomaterial and (B) a substrate having a lower binding ability for the nucleic acid-containing biomaterial than the carrier. ,
a step of contacting the carrier with an aqueous solution containing a nucleic acid amplification reagent containing a primer that enables specific amplification of the specific gene, and subjecting the carrier to conditions for nucleic acid amplification;
A method comprising the step of detecting whether or not said specific gene is contained in said aqueous solution and/or the copy number of said specific gene using said optical means.
(Item 33)
33. The method of item 32, wherein a test sample comprising the nucleic acid-containing biomaterial is provided by contacting the substrate with the nucleic acid-containing biomaterial and then contacting the substrate with the carrier.
(Item 34)
34. A method according to item 32 or 33, wherein the test sample is provided to contain a range of amount of nucleic acid-containing biomaterial.
(Item 34A)
The method of any one of items 32-34, further comprising one or more of the features of items 1-23, 23A, and 24-31.
(Item 35)
A kit for providing a sample for detecting a specific gene contained in a nucleic acid-containing biomaterial and/or the copy number of the specific gene using optical means,
For providing a test sample containing a nucleic acid-containing biomaterial, comprising (A) a carrier capable of binding a nucleic acid-containing biomaterial, and (B) a substrate having a lower affinity for the nucleic acid-containing biomaterial than the carrier. a kit and primers that enable specific amplification of the specific gene;
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
kit.
(Item 35A)
36. The kit of any one of items 35, further comprising one or more features of items 1-23, 23A, and 24-31.
(Item 36)
A system for providing a sample for detecting a specific gene contained in a nucleic acid-containing biomaterial and/or the copy number of the specific gene using optical means,
For providing a test sample containing a nucleic acid-containing biomaterial, comprising (A) a carrier capable of binding a nucleic acid-containing biomaterial, and (B) a substrate having a lower affinity for the nucleic acid-containing biomaterial than the carrier. a kit, primers that enable specific amplification of the specific gene,
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
A system comprising a nucleic acid amplifier.
(Item 36A)
37. The system of any one of items 36, further comprising one or more features of items 1-23, 23A, and 24-31.
(Item 37)
A system for detecting a specific gene contained in a nucleic acid-containing biomaterial and/or the copy number of the specific gene,
For providing a test sample containing a nucleic acid-containing biomaterial, comprising (A) a carrier capable of binding a nucleic acid-containing biomaterial, and (B) a substrate having a lower affinity for the nucleic acid-containing biomaterial than the carrier. a kit, primers that enable specific amplification of the specific gene,
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
A system comprising a nucleic acid amplification device and optical means.
(Item 37A)
38. The system of any one of items 37, further comprising one or more features of items 1-23, 23A, and 24-31.
(Item 38)
A method for detecting a specific gene contained in a nucleic acid-containing biomaterial, comprising:
contacting the test sample containing the nucleic acid-containing biological material with a device containing a non-water-absorbing material, a carrier capable of binding the nucleic acid-containing biological material, and a water-absorbing resin;
a step of contacting the carrier with an aqueous solution containing a nucleic acid amplification reagent containing a primer that enables specific amplification of the specific gene, and subjecting the aqueous solution to conditions for nucleic acid amplification;
A method comprising the step of detecting whether or not said specific gene is contained in said aqueous solution and/or the copy number of said specific gene using said optical means.
(Item 38A)
39. The method of any one of items 38, further comprising one or more of the features of items 1-23, 23A, and 24-31.
(Item 39)
A kit for providing a sample for detecting a specific gene contained in a nucleic acid-containing biomaterial,
A kit for providing a test sample containing a nucleic acid-containing biomaterial, comprising a non-water-absorbing material, a carrier capable of binding a nucleic acid-containing biomaterial, and a device containing a water-absorbing resin, a primer that enables
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
kit.
(Item 39A)
40. The kit of any one of items 39, further comprising one or more features of items 1-23, 23A, and 24-31.
(Item 40)
A system for providing a sample for detecting a specific gene contained in a nucleic acid-containing biomaterial,
A kit for providing a test sample containing a nucleic acid-containing biomaterial, comprising a non-water-absorbing material, a carrier capable of binding a nucleic acid-containing biomaterial, and a device containing a water-absorbing resin, a primer that enables
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
A system comprising a nucleic acid amplifier.
(Item 40A)
41. The system of any one of items 40, further comprising one or more features of items 1-23, 23A, and 24-31.
(Item 41)
A system for detecting a specific gene contained in a nucleic acid-containing biomaterial,
A kit for providing a test sample containing a nucleic acid-containing biomaterial, comprising a non-water-absorbing material, a carrier capable of binding a nucleic acid-containing biomaterial, and a device containing a water-absorbing resin, a primer that enables
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
A system comprising a nucleic acid amplification device and optical means.
(Item 41A)
42. The system of any one of items 41, further comprising one or more features of items 1-23, 23A, and 24-31.

It is contemplated in the present invention that one or more of the features described above may be provided in further combinations in addition to the explicit combinations. Still further embodiments and advantages of the present invention will be appreciated by those skilled in the art upon reading and understanding the following detailed description, if necessary.

INDUSTRIAL APPLICABILITY The present invention enables efficient and easy-to-automate gene amplification, enables efficient, accurate, and highly sensitive detection of specific genes, and enables easy detection of genetic polymorphisms and copy number variations. .

FIG. 1 is a diagram showing the results of analysis using the alcohol predisposition gene (ADH1B) as an example. Alcohol predisposition gene (ADH1B) using an applicator (Trust ^CatcherTM ) (bottom left), angular (gamma-ray sterilized) swab (top left) or round (unsterilized) swab (top right) (CleanTip® Swabs, Texwipe) Then, oral mucosa cells were collected, pressed onto water-soluble paper, transferred, dried, and amplified. In each figure, the X-axis indicates allele X (allele 2) and the Y-axis indicates allele Y (allele 1). Diamonds indicate A (pc), triangles indicate A/G (pc), circles indicate G (pc) and squares indicate distilled water (nc). X indicates samples with weak signals that could not be determined. Distilled water was used as a negative control (nc) which generally gave no signal. A positive control (pc) is recombinant DNA in which the detection gene region of each genotype is incorporated into plasmid DNA. Amplification conditions are heat denaturation: 40 cycles of 50°C, 2 minutes → 95°C, 10 minutes, 95°C, 15 seconds → 60°C, 1 minute. When saliva was collected with a dropper, signals from several samples (X) appeared near distilled water and were difficult to distinguish. On the other hand, all of the other three are considered to have sufficient discriminative power. In the method using a dropper, the amount of DNA is not constant (both signals flow in the direction of distilled water). FIG. 2 is a diagram showing the results of analysis using the alcohol predisposition gene (ALDH2) as an example. Using an applicator (Trust Catcher ^TM ) (bottom) or a horn (gamma-ray sterilized) swab (top) for the alcohol predisposition gene (ALDH2), oral mucosa cells were collected, pressed on water-soluble paper, and transferred to cells. The results of drying and subjecting this to amplification are shown. In each figure, the X-axis indicates allele X (allele 2) and the Y-axis indicates allele Y (allele 1). Diamonds indicate G (pc), triangles indicate G/A (pc), circles indicate A (pc) and squares indicate distilled water (nc). X indicates samples with weak signals that could not be determined. Amplification conditions are heat denaturation: 40 cycles of 50°C, 2 minutes → 95°C, 10 minutes, 95°C, 15 seconds → 60°C, 1 minute. There were no samples that could not be judged, and all were considered to have sufficient discriminative power. Distilled water was used as a negative control (nc) which generally gave no signal. A positive control (pc) is recombinant DNA in which the detection gene region of each genotype is incorporated into plasmid DNA. As such, ALDH2 is expected to exhibit the same tendency as ADH1B, so the following experiments were performed using ADH1B as a representative example. FIG. 3 shows the results of comparison using various qualitative filter papers and water-soluble papers targeting ADH1B, and samples obtained by dripping and drying saliva collected with an applicator (Trust Catcher ^™ ). Saliva (oral mucosal cells) collected with an applicator (Trust Catcher ^™ ) was dropped and dried, cut into 4 mmφ, suspended in 50 µL LDW, and 2 µL of which was heat-treated at 95°C for 5 minutes was used. Water-soluble paper is shown to be a very good member. In each figure, the X-axis indicates allele X (allele 2) and the Y-axis indicates allele Y (allele 1). ▲ indicates G/A and □ indicates distilled water. Distilled water gives no signal, neither G nor A). The table below shows various specifications for filter paper and water soluble paper. The amount of cells retained was almost constant regardless of the paper quality of the water-soluble paper. Although 60 MDP was most preferable from the viewpoint of operability, it is not limited to this. In the ADVANTEC filter paper column, the filter papers of No. 4A has less cell retention. Fig. 3A shows ADH1B as a target, Kimwipe (Nippon Paper Crecia), Kimtowel (Nippon Paper Crecia), newspaper (Yomiuri Shimbun, using the unprinted part) and tissue paper, and collected with an applicator (Trust Catcher ^TM ). FIG. 10 is a diagram further showing the results of comparison using a sample obtained by dripping and drying saliva that has been treated. Saliva (oral mucosal cells) collected with an applicator (Trust Catcher ^™ ) was dropped and dried, cut into 4 mmφ, suspended in 50 µL LDW, and 2 µL of which was heat-treated at 95°C for 5 minutes was used. The X-axis shows allele X (allele 2) and the Y-axis shows allele Y (allele 1). Black triangles indicate G/A, and open squares indicate distilled water. Distilled water was used as a negative control (nc) which generally gave no signal. A positive control (pc) is recombinant DNA in which the detection gene region of each genotype is incorporated into plasmid DNA. FIG. 3B shows the difference in results with and without heat treatment at 95° C. for 5 minutes after sampling with various substrates (with swabs as substrates, varying the number of rubs). It is a diagram. The X-axis shows allele X (allele 2) and the Y-axis shows allele Y (allele 1). FIG. 3C shows the difference in amplification curves with and without heat treatment at 95 ° C. for 5 minutes after sampling using various substrates (with swabs as substrates, changing the number of rubs). FIG. 4 is a diagram showing; The horizontal axis is the cycle number, and the vertical axis is the fluorescence signal (ΔRn) corresponding to the amplified PCR product. FIG. 3D is a diagram excluding the data using newspaper and Kimwipes as base materials from FIG. 3B, and shows the results of an experiment conducted by changing the number of times the swab was rubbed in the oral cavity. The discriminative power of genotypes does not depend on the number of rubs in the oral cavity. FIG. 3E is a diagram excluding the data using newspaper and Kimwipe as substrates from FIG. 3C, and showing the difference in the amplification curve in the experiment performed by changing the number of times the swab was rubbed in the oral cavity. The horizontal axis is the cycle number, and the vertical axis is the fluorescence signal (ΔRn) corresponding to the amplified PCR product. The discriminative power of genotypes does not depend on the number of rubs in the oral cavity. FIG. 3F shows the results of real-time PCR amplification using samples prepared using different swab-like substrates. The X-axis shows allele X (allele 2) and the Y-axis shows allele Y (allele 1). Diamonds indicate A (positive control = pc), triangles indicate G/A (positive control = pc, sample), circles indicate G (positive control = pc), squares indicate distilled water (negative control = nc). indicates Using swab (polyurethane), swab short foam type (polyurethane), tip of applicator (Trust Catcher ^TM ) (polyvinyl alcohol (PVA)), and cotton swab (cotton) as substrates for oral mucosa cell collection. Cells were harvested and transferred by pressing onto water-soluble paper. The results indicated that good genotype discrimination was possible for any base material. FIG. 3G shows the difference in genotyping for samples prepared by swabbing with and without heating and between suspension and supernatant. The X-axis shows allele X (allele 2) and the Y-axis shows allele Y (allele 1). FIG. 3H shows the results of genotyping of 6 subjects using swabs and applicators (Trust Catcher ^™ ) as substrates. The X-axis shows allele X (allele 2) and the Y-axis shows allele Y (allele 1). Diamonds indicate samples rated A, triangles indicate samples rated A/G, and circles indicate A/G positive controls (pc). Without wishing to be bound by theory, quantitation was more accurately achieved with swabs where the surface area remained nearly constant, whereas the applicator deformed its shape upon application and was absorbed internally. Swabs appear to be more quantitative than applicators because of the exudation of sample (also called volume effect). This is thought to be due to the volume effect sometimes exceeding the detection limit in the shape of an applicator. FIG. 4 shows the results of melting curve analysis of the angiotensin-converting enzyme (ACE) gene defect by endpoint analysis of real-time PCR reaction using SYBR (registered trademark) Green. The upper row is a graph showing individual melting curve analysis. They are I/I, I/D and D/D from the left, and the Tm values are shown in the figure. The bottom row shows, from the left, 3 types of superimposed melting curve analysis, 3 types of superimposed amplification curves, and amplification curves for all 12 subjects. It was shown from the melting curve that gene polymorphism analysis can be performed without any problems. The amplification curve Ct value is 23.7 cycles ± 2.6, which means that there is no problem in terms of sensitivity, and even though DNA extraction is cut, ideal detection is achieved. This is proof of that. FIG. 4A shows detection of ACE genotypes by automated microchip electrophoresis with MultiNA (MCE-202, Shimadzu). Lane 1: marker, lane 2: ACE D/D (190 bp), lane 3: ACE I/I (490 bp), lane 4: ACE I/D (490, 190 bp), lane 5: DW, LM: lower marker , UM: upper marker. FIG. 5 shows that the copy number variation of CYP2D6, which is a cytochrome 450-metabolizing enzyme, can be quantitatively detected by the TaqMan (registered trademark) probe method without DNA extraction and purification. It is the data which shows a result. The upper part shows, from the left, that 1 copy can be calculated when ΔΔCt=approximately 1, and 2 copies can be calculated when ΔΔCt=approximately 2. The lower part shows the amplification curve when 4 specimens of 1 copy from the left are superimposed (each Ct value is CTP2D6: 26.9±0.9 cycles, RNaseP: 28.2±0.5 cycles). Amplification curves (each Ct value is CTP2D6: 25.7±2.3 cycles, RNaseP: 27.6±1.8 cycles) when 2 copies of 10 samples are superimposed are shown. Using the RNase P gene without copy number polymorphism as a reference, the copy number polymorphism of CYP2D6 was calculated from the difference in Ct values (ΔΔCt). The amplification curve Ct values of all 14 specimens are CTP2D6: 26.0 ± 2.3 cycles, RNaseP: 27.8 ± 1.8 cycles, there is no problem in terms of sensitivity, and DNA extraction is cut. This is proof that ideal detection is possible despite the fact that The amplification curve Ct value is CYP2D6: 26.0 cycles ± 2.3 for the two-copy allele, and the reference RNaseP: 27.8 cycles ± 1.9, so there is no particular problem in terms of sensitivity. It has been shown that it is possible to determine FIG. 6 is a schematic diagram showing a calculation method for detecting copy number variation using the TaqMan (registered trademark) PCR method. The lower left rectangular figure is a bar graph showing the results of copy number polymorphism determination for each specimen. The short one can be separated into 1 copy, and the longer one can be separated into 2 copies, and the copy number variation can be determined. The lower right figure is a typical amplification curve. The lower right graph shows an example of calculating copy number variation from the Ct value of the amplification curve. FIG. 7 shows the shape and usage of swabs (polyurethane swabs are typically used) used in the present invention. (1) Place the sponge part of the swab in your mouth, and if necessary, (2) rub one side of the sponge on the mucous membrane on the inner side of your right cheek in a circular motion more than 15 times. It has been shown that similar results are obtained. (3) On the other side, the left inner side of the cheek is carefully rubbed 15 times or more, but it has been shown that similar results can be obtained without rubbing. (4) Rub the back of the tongue with the same sponge as needed, but has been shown to produce similar results without rubbing. Next, press both sides of the swab against the water-soluble paper as shown in the middle row. Further, as exemplified in the lower part, it is sufficiently dried (typically, 30 minutes or longer). FIG. 8 is a schematic diagram of sample preparation using water-soluble paper. At the top is a bird's-eye view from above. A piece of water-soluble paper is placed in the center with a standard size, here a diameter of 20 mm. The figure seen from the side is the second figure from the top. Thus, the device of the present invention is composed of two layers, and the double-sided adhesive tape as a preferred embodiment is arranged between them. The upper layer is plastic in material and the lower layer is water-absorbent polymer. The top layer of plastic is perforated and water-soluble paper is placed. Moisture is absorbed by using a water-absorbing polymer (eg, Teijin Peroasis, etc.). It is preferable that the double-sided adhesive tape can be peeled off after the fact. This allows the water-absorbing polymer to be peeled off. After that, the upper layer holding the water-soluble paper is dried (third row from the top=middle) and then placed on the cutter table. Using a biopsy trepan available from Kaijirushi, etc., a water-soluble paper of a predetermined size is hollowed out. FIG. 9 is an illustration of a scheme of the direct TaqMan (registered trademark) PCR method using an oral mucosa cell sample. Add 50 μL of water (distilled water=DW) and heat at 95° C. for 5 minutes. Centrifuge and store in refrigerator (4°C). 1 μL of this extract is added to TaqMan (registered trademark) PCR reaction solution (10 μL system) and reacted. On the left is a schematic diagram. FIG. 10 is a schematic diagram of sample preparation when using an applicator (Trust Catcher ^™ ). Samples can be prepared by placing the sponge portion in the mouth and sucking saliva based on the standard usage of the device. The sponge is then inverted and sampled by placing 3 drops into the application hole (6 mm diameter). FIG. 11 details sample preparation when using an applicator (Trust Catcher ^™ ). The top is a bird's-eye view from above. A case with a diameter of 6 mm is shown. The second from the top shows a side view of the first state. Similar to FIG. 8, the device of the present invention is composed of two layers with a double-sided adhesive tape as a preferred embodiment between them. The upper layer is plastic in material and the lower layer is water-absorbent polymer. The top layer of plastic is perforated and water-soluble paper is placed (dropped or added) into the water. Moisture is absorbed by using a water-absorbing polymer (eg, Teijin Peroasis, etc.). It is preferable that the double-sided adhesive tape can be peeled off after the fact. This allows the water-absorbing polymer to be peeled off. After that, the upper layer holding the water-soluble paper is dried (third row from the top = center), and then it is directly placed in a reaction tube, for example, 100 μL of distilled water is added and reacted at 95° C. for 5 minutes. After this, the reaction can proceed as shown in FIG. FIG. 12 is a schematic diagram showing the sampling process. The left panel shows examples of specialized polyurethane swabs. The water-soluble paper is designed with a diameter of 6 mm and constitutes the sampling plate. Allow this to dry for 5 minutes, flip it over, and use a pick stick (pick-up stick) as shown in the left panel so that it can be placed in a TaqMan® PCR reaction preparation tube and placed immediately. can be analyzed by real-time PCR. Figure 13 shows a schematic scheme of the present invention. FIG. 13A shows a schematic scheme of the conventional method. FIG. 14 is another embodiment of the invention. The shape of the sampling sheet and double-sided tape are described in the left panel, and the schematic diagram of the actual usage is described on the right side. At the bottom, examples of products using the carrier of the present invention are shown. FIG. 15 shows detection of genes in samples prepared using newspaper as a carrier. The X-axis shows ADH1B allele X (allele 2) and the Y-axis shows ADH1B allele Y (allele 1). Filled triangles ▴ indicate signals derived from saliva samples from various samplings, and filled circles are serially diluted positive controls. FIG. 16 shows detection of genes in samples prepared using qualitative filter paper as a carrier. The X-axis shows ADH1B allele X (allele 2) and the Y-axis shows ADH1B allele Y (allele 1). Filled triangles ▴ indicate signals derived from saliva samples from various samplings, and filled circles are serially diluted positive controls. FIG. 17 shows detection of genes in samples prepared using water-soluble paper as a carrier. The X-axis shows ADH1B allele X (allele 2) and the Y-axis shows ADH1B allele Y (allele 1). Filled triangles ▴ indicate signals derived from saliva samples from various samplings, and filled circles are serially diluted positive controls. FIG. 18 is a diagram showing the results of an experiment conducted by changing the number of transfers from the swab to the water-soluble paper. The X-axis shows ADH1B allele X (allele 2) and the Y-axis shows ADH1B allele Y (allele 1). Filled triangles ▴ indicate signals derived from saliva samples from various samplings, and filled circles are serially diluted positive controls. FIG. 19 is a diagram showing the results of a quantitative test of a sample released into water from water-soluble paper using saliva serially diluted in a 10-fold dilution series. The X-axis shows ADH1B allele X (allele 2) and the Y-axis shows ADH1B allele Y (allele 1). Filled triangles ▴ indicate signals derived from saliva samples from various samplings, and filled circles are serially diluted positive controls. Assuming a linear function, the regression rate is calculated to be 0.95 or more. FIG. 19A shows a calibration curve based on the amplification curve of the positive control in the quantitative test of the sample released from water-soluble paper into water. The vertical axis is the Ct value and the horizontal axis is the copy number. The copy number of the positive control can be spectroscopically calculated from the purified DNA (copy number per μL). Linearity is shown for both alleles 1 and 2. Assuming a linear function, the regression rate is calculated to be 0.95 or more. FIG. 19B shows the quantitative test of the sample released into water from water-soluble paper using saliva serially diluted in a 10-fold dilution series. Calculated results are shown. When dropped onto water-soluble paper, linearity is shown up to 1000-fold dilution. FIG. 20 is a schematic diagram of a case where a sample is adhered to the sampling sheet of the present invention and then transported in a drying box. As shown in the right figure of Fig. 20, if a part of the sampling sheet can be folded but not completely folded, that part will act as a spacer and the sample on the sampling sheet will adhere. It can be transported in a box (see the left figure in Fig. 20) while keeping the exposed part from touching other parts. Figure 21 compares the performance of functional water-soluble paper (60MDP, Nippon Paper Papilla) with qualitative filter paper (ADVANTEC No. 1) and Whatman® 903 protein saver card by SYBR® Green real-time assay method. It is a figure which shows the result of performing performance evaluation. From the left, black line: functional water-soluble paper (60MDP, Nippon Paper Papilia), blue line: Whatman (registered trademark) 903 protein saver card, red line: qualitative filter paper (ADVANTEC No. 1), green line: negative control (distillation water). FIG. 22 shows the results of ADH1B and ALDH2 genotyping. FIG. 23 shows the results of ACE genotyping by melting curve. FIG. 24 shows a performance comparison of water-soluble paper against dry saliva samples. FIG. 25 shows the performance comparison of water-soluble paper on dried blood samples.

Hereinafter, the present invention will be described while showing the best mode. It should be understood that throughout this specification, expressions in the singular also include the concept of the plural unless specifically stated otherwise. Thus, articles in the singular (eg, “a,” “an,” “the,” etc. in the English language) should be understood to include their plural forms as well, unless otherwise stated. Also, it should be understood that the terms used in this specification have the meanings commonly used in the relevant field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification (including definitions) will control.

Definitions of terms and/or basic technical content particularly used in the present specification will be described below as appropriate.

As used herein, the term "nucleic acid-containing biomaterial" refers to any biomaterial containing a specific target nucleic acid, and includes biological tissues/cells (e.g., plant and animal cells), bacteria, viruses, and the like. Examples of "samples containing nucleic acid-containing biomaterials" include nasal discharge, nasal swabs, conjunctival swabs, pharyngeal swabs, sputum, feces, blood, serum, and plasma. , cerebrospinal fluid, saliva, urine, sweat, milk, semen, oral swab, interdental swab, wet cerumen, vaginal swab, and biological samples or clinical samples such as cell tissue, as well as foods. . A test sample may comprise a sample containing RNA selected from the group consisting of cells, fungi, bacteria and viruses. A gene to be amplified or a specific gene to be detected may be either DNA or RNA. In this case, in the present invention, the RNA present in the test sample can be directly amplified by directly adding the test sample supported on the water-decomposable carrier to the reaction solution to carry out the RNA amplification reaction. can. Here, "direct addition" means that a process of extracting RNA from a test sample containing this RNA is unnecessary prior to RNA amplification.

As used herein, the term "capable of binding nucleic acid-containing biomaterials" refers to a carrier or substrate, any carrier or substrate capable of binding nucleic acid-containing biomaterials (typically cells) Say. As used herein, whether or not "a nucleic acid-containing biomaterial can be bound" can be measured under the following conditions.

For example, it can be determined by the following tests.

(Nucleic acid-containing biomaterial binding test)
1) Prepare a punch carrier (newspaper, qualitative filter paper No. 1, water-soluble paper 60 MDP) with a certain area (for example, 6 mmΦ (diameter)),
2) drop a certain amount of cell-containing solution (for example, 10 μL of saliva) onto the carrier and air dry for 1 hour;
3) then immersing the carrier in water;
4) After that, the number of cells released in water within a certain period of time (0 to 60 minutes, 0 minutes, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 60 minutes) was measured by real-time PCR from the ADH1B control plasmid. and quantify. After that, heat treatment is performed at 95° C. for 5 minutes to determine how many cells adhere to the carrier or substrate, and whether the cells can be released when immersed in water is determined by the “retention force capable of binding nucleic acid-containing biomaterials”. be able to.

In this case, the carrier or substrate is "capable of binding nucleic acid-containing biomaterials" if it is calculated to liberate a sufficient number of cells (approximately 1×10 ¹ copy cells) to perform genetic testing in water. can be determined. The term "X copy cells" means "the number of cells that can release X copies of a specific gene".

As the condition of 3), a heating step (for example, heat treatment at 95° C. for 5 minutes) or a boiling step can be assumed. In that case, a step of boiling after soaking in water can be included.

In this case, in this specification, whether or not the carrier or base material "can bind to a nucleic acid-containing biomaterial" is determined at least for genetic testing in water when the heating step or boiling step in 3) above is included. If it is calculated to liberate a sufficient number of cells (approximately 1×10 ¹ copy cells), it can be determined to be “capable of binding nucleic acid-containing biomaterials”. In this case, when specifically distinguished, it can be described as "capable of being released under heating conditions" or "capable of being released under boiling conditions" and "capable of binding nucleic acid-containing biomaterials". In 3), the carrier or base material "capable of binding nucleic acid-containing biomaterials" without including heating or boiling conditions is usually "releasable under heating conditions" or "releasable under boiling conditions" or "nucleic acid-containing It is a carrier or substrate capable of binding biomaterials. In addition, even if it is not determined that "the nucleic acid-containing biomaterial can be bound" even if the heating condition or boiling condition is not included in 3), in some cases, "be able to be released under heating conditions" or "under boiling conditions It may be a carrier or substrate that is releasably "capable of binding nucleic acid-containing biomaterials." Therefore, in general, the present invention uses a carrier or substrate that is "capable of being released under heating conditions" or "capable of being released under boiling conditions" and "capable of binding nucleic acid-containing biomaterials", but in preferred embodiments , a carrier or base material that is “releasable under heating conditions” and “can bind nucleic acid-containing biomaterials” can be used, and more preferably, in 3), “nucleic acid-containing biomaterials” can be used without using heating conditions or boiling conditions. A carrier or substrate capable of binding"can be used.

In another preferred embodiment, it is preferable to conduct a similar experiment with three or more concentrations, plot each point, and then linearly regress. This is because quantification is more guaranteed in the case of such linear regression. In this case, the regression rate is usually 0.6 or more, preferably 0.7 or more, preferably 0.8 or more, preferably 0.9 or more, preferably 0.95 or more, preferably 0.98 or more, preferably 0 .99 or higher. In the present invention, it may be preferable to use materials with a regression rate of 0.8 or greater, more preferably 0.9 or greater, even more preferably 0.95 or greater, 0.98 or greater, or 0.99 or greater, Such regression rates may vary depending on the quality of quantification sought. Quantitative confirmation can also be confirmed by plotting the amplification products of the two genes at 3 or more concentrations, as shown in FIG. 19, and confirming their linear relationship.

It should be noted that the newspaper and filter paper used in the examples do not liberate a sufficient number of cells (approximately 1×10 ¹ copy cells) for genetic testing in water only by immersion in water (therefore, in this case, SNP determination is not possible). ) releases 10 ² copies under the heating conditions of heat treatment at 95° C. for 5 minutes, indicating that the cells are liberated.

In the case of the water-soluble paper used in the examples, 10 ² to 10 ³ copies of cells were released into the water simply by immersion in water, and 10 ⁴ copies of genomic DNA were released by heat treatment at 95°C for 5 minutes. It is shown. Water-soluble paper captures about 100 times more oral mucosal cells than newspaper and filter paper when swabs are used.

In an illustrative example, as shown in FIG. 19, from the Ct value of the real-time PCR amplification curve of each carrier heat-treated at 95° C. for 5 minutes, 10 ² copies of cells were obtained for newspaper and filter paper, and 10 ⁴ copies for water paper. A punch with a constant area of 6 mmφ was immersed in a constant amount of 200 μL of water, and 5 μL of the punch was used for DNA quantification by real-time PCR. It is

As used herein, the term “substrate having a lower nucleic acid-containing biomaterial binding capacity than the carrier” means that when the carrier and the substrate are compared, the nucleic acid-containing biomaterial (e.g., cells) binding capacity is lower than that of the substrate. is lower than the carrier, and when the nucleic acid-containing biomaterial is carried on the substrate, when the substrate is brought into contact with the carrier, a certain amount of the nucleic acid-containing biomaterial (e.g., cells) is preferably Refers to a combination of substrate and carrier such that substantially all nucleic acid-containing biomaterials (eg, cells) are transferred to the carrier. When a given carrier is given, it can be confirmed, for example, by the following test whether or not an arbitrary substrate has a lower ability to bind nucleic acid-containing biomaterials than the carrier.

In the present specification, for example, it is possible to determine a "substrate having a lower nucleic acid-containing biomaterial binding capacity than the carrier" by methods such as the following binding capacity comparison method (1) and binding capacity comparison method (2). can.
(Binding ability comparison method (1))
1) providing a carrier and a substrate;
2) placing a certain amount of cell-containing solution on the substrate for several minutes (eg, 5 minutes);
3) then contacting the substrate and the carrier for a certain period of time (for example, 5 seconds);
4) After that, measure how much cells or nucleic acids remain in the base material and the carrier (in the case of nucleic acids, for example, using the above-described quantitative PCR)), and with the measurement results, more than half of the nucleic acid-containing biomaterials is transferred to the carrier, preferably substantially all of the nucleic acid-containing biomaterial is transferred, the substrate is determined to be "a base material having a lower ability to bind the nucleic acid-containing biomaterial than the carrier". Carriers or substrates commonly used in the present invention are those "capable of binding nucleic acid-containing biomaterials."

(Binding capacity comparison method (2))
(1) Providing a carrier and a substrate.
(2) A sample containing a given amount (eg, 10 μL) of nucleic acid is brought into contact with the carrier.
(3) This carrier is brought into contact with a substrate candidate to be tested for a certain period of time.
(4) After a certain period of time has elapsed, cut off a certain area (for example, 6 mmΦ (diameter)) of the surfaces of the carrier and the substrate candidate that were in contact with each other.
(5) The amounts of nucleic acids contained in the cut sections are measured (for example, using quantitative PCR as described above) and compared.
(6) If an amount of nucleic acid less than the amount of nucleic acid extracted from the test carrier is found in the candidate base material, the candidate base material is adopted as the base material used in the present invention.

Here, it can be said that there is a relative relationship between a suitable substrate and a carrier.

As used herein, the term "carrying a nucleic acid-containing biomaterial without binding" refers to, for example, a case in which substantially all of the nucleic acid-containing biomaterial is transferred in the above test.

In the present specification, the nucleic acid-containing biomaterial in a "certain range amount" refers to an amount that avoids confusion with other results in the measurement or analysis performed after nucleic acid amplification in the present invention. For example, within 64-fold (+6 cycles) to 1/64 (-6 cycles) of the reference amount, and X is the fold difference that can be distinguished when the copy number differs by 1 in the nucleic acid amplification, For example, 32 times to 1/32 times, 16 times to 1/16 times, 8 times to 1/8 times, 4 times to 1/4 times, 10 times the square root to 10 times the square root of 1, 3 times 1/3, 1.7 to 1.8 times to 1.7 to 1.8 times, and the like. Alternatively, it is within X times to 1/X of the reference amount, and ^X2 is the difference in fold that can be distinguished when the copy number differs by 1 in the nucleic acid amplification. When the copy number is 0, no amplification of the target gene is obtained. When the copy number is 1, the copy number of the reference DNA (generally using RNaseP) is 2, so the copy number is converted based on the difference in Ct value. In the case of CYP2D6 exemplified in the examples, ΔCt = 1 when the copy number is 1, ΔCt = 2 when the copy number is 2, and ΔCt = 2.3 to 2.6 when the copy number is 3. , but not limited to these.

As used herein, the expression that the carrier "permits nucleic acid amplification when placed in water" means that the carrier does not inhibit the nucleic acid amplification reaction when placed in water or an aqueous solution (including amplification reagents, etc.), or Inhibition of the nucleic acid amplification reaction can be avoided by adding an additional specific reagent (for example, about 10% of KOD FX Neo buffer attached to KOD FX Neo (Toyobo Life Science)).

As used herein, the expression that the carrier "allows optical detection" means that optical detection (including the naked eye) when placed in water or an aqueous solution (including amplification reagents, etc.) ) is possible.

As used herein, the term "water-decomposable carrier" refers to a carrier that has the property of being decomposed upon contact with water and dissolved or dispersed in water. Whether or not the water is decomposable can be determined, for example, by contacting 0.05 to 0.1 mg of the carrier with 10 μl of water (reaction solution), and measuring the turbidity of the water (reaction solution) at 660 nm with Bio- Water decomposability is considered to be 0.4 or less when measured using the RAD Smart Spec™ 3000. Specific examples of such water-decomposable carriers include 60MDP (manufactured by Nippon Paper Papylia Co., Ltd.), 30MDP (manufactured by Nippon Paper Papylia Co., Ltd.), 120MDP (manufactured by Nippon Paper Papylia Co., Ltd.), and 30MDP-S (manufactured by Nippon Paper Papylia Co., Ltd.). , MDP series products such as 60MDP-S (manufactured by Nippon Paper Papylia Co., Ltd.), 30CD-2 (manufactured by Nippon Paper Papylia Co., Ltd.), 60CD-2 (manufactured by Nippon Paper Papylia Co., Ltd.), 120CD-2 (manufactured by Nippon Paper Papylia Co., Ltd.) Water-dissolvable paper such as the CD-2 series, water-dissolvable paper (manufactured by Tokushushi Shoji Co., Ltd.), agarose gel dried and made into a film, gelatin, collagen, etc. dissolved by heating and dried and made into a film etc. Among them, in the reaction solution used for gene amplification or specific gene detection, the water-dissolvable carrier disperses uniformly during the reaction, further precipitates, and becomes a state that does not easily interfere with physical manipulation and optical detection. It is preferable to use water-disintegrable paper as the. Also, in another preferred embodiment, those that allow for optical detection are preferred. As used herein, "water-disintegrable paper" may also be referred to as "water-disintegrable paper" or "water-soluble paper," and these terms are used interchangeably.

Ordinary paper is mainly made of vegetable fibers (eg, cellulose) and is made of intertwined fibers. Hydrogen bonds are formed between these fibers by the OH groups of cellulose, but these bonds are released in water, so paper generally loses its physical strength due to water, and the fibers disperse in water over time. do. Water soluble paper is paper manufactured to further enhance such water soluble properties. Water-soluble paper is formed by forming paper using a derivative obtained by introducing a hydrophilic functional group into cellulose, such as a water-soluble cellulose derivative such as hydroxyethyl cellulose or carboxymethyl cellulose (CMC), as a skeleton. It can be produced by making the cellulose itself easily soluble in water. For example, the CMC can be Sunrose (registered trademark) (Nippon Paper Industries) and the like. CMC has a carboxymethyl group substituted for the OH group and is solubilized by the polarity of the carboxyl group. Glucose, which is a unit of cellulose, has three OH groups, and when 0.3 or more OH groups on average per glucose unit are substituted with carboxymethyl groups, water solubility begins to occur. Such water-soluble cellulose derivatives may be obtained by substituting various ratios of OH groups in the cellulose fibers. In addition, such water-soluble cellulose derivatives can be included in paper fibers at any ratio. Alternatively, water-soluble paper can be produced by binding fibers of paper using a water-soluble binder instead of physical entanglement, whereby the binding between fibers is dissolved in water. Such water-soluble properties of paper are described in Textile and Industry Vol. 1 1968 No. 10, pp. 604-608, Toshiharu Emae, Paper and Paper Technology Association 58(8), Papyrus, 105-109 (2004), etc., and is well known to those skilled in the art.

Examples of water-soluble paper include paper prepared by blending fibrous carboxymethyl cellulose (fibrous CMC) obtained by carboxymethylating cellulose fibers with pulp, making paper, and then converting CMC into its alkali metal salt with an alkali agent. The water dispersion solubility of this paper can be adjusted by adjusting the amount of carboxymethyl groups and the degree of substitution with alkali metal salts. The fibrous CMC alkali metal salt has the property of swelling when immersed in water and then dissolving. When water-soluble paper containing a fibrous CMC alkali metal salt is immersed in water, the fibrous CMC alkali metal salt first swells and then dissolves, thereby binding the pulp fibers that are in contact with water. It is considered that the water-soluble paper exhibits water-dispersible solubility.

Such water-soluble paper (water-dissolvable paper) can be used as a preferred carrier in the present invention. For example, JP-A-2006-180983, JP-A-2013-185259, JP-A-2006-2296, JP-A-2012-41649, JP-A-6-138121, JP-A-10-36776, JP-A-11-279995 Water-soluble papers described in the literature, such as water-soluble papers, or water-soluble papers produced by the methods described can also be used in the present invention. Commonly available commercially available tissues (water soluble) and paper such as those used for goldfish scooping poi can also be used.

Water-dissolvable carriers can be produced using water-soluble materials other than so-called paper mainly composed of cellulose. The water-soluble material may be a water-soluble polymer, such as starch (such as corn starch or potato starch), mannan, pectin, agar, alginic acid, vegetable gums (xanthan gum, guar gum), dextran, pullulan, glue or gelatin. natural macromolecules such as proteins such as Alternatively, the water-soluble material can be a synthetic polymer such as sodium polyacrylate, polyacrylamide, polyvinyl alcohol, polyethyleneimine, polyethylene oxide, polyvinylpyrrolidone, and the like. Water-degradable materials such as those described above can be used as long as they have desirable binding properties with nucleic acid-containing biomaterials as carriers described herein (for example, higher binding capacity for nucleic acid-containing biomaterials than for substrates). A water-disintegratable carrier can be used.

These materials were subjected to a "nucleic acid-containing biomaterial binding test" to confirm that they "can be released under heating conditions" or "can be released under boiling conditions" and "can bind nucleic acid-containing biomaterials". In 3), preferably, a carrier or base material confirmed to be "capable of binding nucleic acid-containing biomaterials" without using heating conditions or boiling conditions can be used. In addition, even if the quantification is not sufficient when quantifying without using the heating condition or the boiling condition in 3), the quantification is exhibited in "can be released under heating conditions" or "can be released under boiling conditions". can also be used.

By using such a water-decomposable carrier, the water-decomposable carrier carrying the test sample is brought into contact with the reaction liquid, whereby the water-decomposable carrier is hydrolyzed in the reaction liquid. Since the water-degradable carrier is provided with a sample containing a nucleic acid-containing biomaterial in a certain amount from the base material, as a result, the copy number of a specific gene can be specified and/or simply by observing the state of amplification. Presence or absence can be detected. In the past, this was achieved by drying a liquid substance on a water-dissolvable carrier, but copy number variation was not considered, and therefore, any substance capable of gene amplification can be used. They were used indistinguishably.

In addition, it was found that even when blood or saliva dried on a water-dissolvable carrier is used as a test sample, the number of cells contained in saliva or the like is not always constant. That is, it was found that about 10% of the samples required re-experimentation because the number of oral mucosa cells in saliva varied and specific detection was not possible. The reason for this is thought to be that, in amplification reactions such as PCR, which are commonly used, non-specific amplification becomes pronounced after 30 cycles, making it difficult to detect specific genes or significantly lowering reliability. Therefore, in the conventional method, it is not possible to reliably detect a specific gene at more than 30 cycles, and it is not possible to detect copy number variation without purification. The results were unreliable (verification experiment and validation of SNP typing analysis method for alcohol metabolism-related genes ADH1B and ALDH2 using dried saliva. Shigenori Murata et al., Clinical Pathology, Vol. 63, No. 11, p. 1253). ).

When transporting or storing samples containing nucleic acid-containing biomaterials, such as blood samples, the presence of water in the sample may cause cell degradation by proteases, peptidases, etc., and nucleic acids such as DNase, RNase, etc. Degradative enzymatic reactions degrade the sample. Enzyme reaction can be suppressed by using a carrier with appropriate water absorption that can eliminate the water in the sample. becomes.

After attaching a sample to a sampling sheet or the like containing the carrier of the present invention, it can be transported in a drying box. If a part of the sampling sheet is allowed to be folded, but not completely folded, that part acts as a spacer, and the sample-attached part of the sampling sheet touches the other parts. It can be transported in a box while keeping it safe. As such an example, a mode as described in FIG. 20 can be exemplified. Such a sampling sheet and box combination can be manufactured and provided as a sampling kit.

Also, the sampling sheet or the like containing the carrier of the present invention can be placed in a sealed bag (eg, made of plastic) (Fig. 14). More specifically, for the sealed bag, for example, one provided by Seisan Nihon Co., Ltd., etc. can be used, and in particular, nylon (NY) / polyethylene (PE) that can use a desiccant can be used. A flat bag NY type (LZ), which is a laminated bag with a zipper, is desirable. Examples of sealed bags can be found at http://www.seinichi.co.jp/product_info/search_list.html?pa=2&y_id=14, etc. Flat bag NY type (LZ) (NY/PE chuck three-sided bag with zipper), flat bag AL type (AL) (PET/AL/PE three-sided bag with zipper), flat bag barrier NY type (BY) (barrier NY/PE type three-sided bag with zipper), flat bag white pouch (R ) AL type (PET/AL/PE three-sided bag with zipper white printing), stand pack AL type (AL) (PET/AL/PE stand type), stand pack kraft type (KR) (kraft paper/AL/NY/PE stand type) and the like, but are not limited thereto. At this time, a desiccant (silica gel, quicklime (calcium oxide), calcium chloride, deciclay, synthetic zeolite, molecular sieve, aluminum oxide, etc.) can be added together. This promotes drying of saliva and the like after sampling, further enhances the performance of the water-soluble carrier, and enables more stable genetic analysis. In one embodiment, the desiccant may be provided in sheet form. In another embodiment, the desiccant can be placed in parallel with the sampling sheet. A desiccant may be provided over the portion of the sampling sheet where the samples are provided.

Further, for example, when water-soluble paper (120 MDP) is used as a carrier, it has sufficient thickness and/or grammage, so it is not necessary to fix the water-soluble paper on the mounting paper when producing a product or kit. Therefore, it is therefore possible to hollow out the carrier directly for analysis. In addition, since it has sufficient thickness and/or basis weight, it is possible to cut out a certain area by simply extruding without using a punch or the like by making a cut in a certain area in advance (Fig. 14). Therefore, for example, the water-soluble paper used in the present invention preferably has a basis weight of more than 60 g/m ² , such as about 65 g/m ² or more, about 70 g/m ² or more, about 75 g/m ² or more, about 80 g. /m ² or more, about 85 g/m ² or more, about 90 g/m ² or more, about 95 g/m 2 or more, about 100 g/m ^{2 or} more, about 105 g/m ^{2 or} more, about 110 g/m ² or more, about 115 g/m m ² or more, such as about 120 g/m ² or more. For example, the preferred thickness of the water-soluble paper used in the present invention is more than 110 μm, for example, about 115 μm or more, about 120 μm or more, about 125 μm or more, about 130 μm or more, about 135 μm or more, about 140 μm or more, about 145 μm or more, It may be about 150 μm or more, about 155 μm or more, about 160 μm or more, about 165 μm or more, about 170 μm or more, about 175 μm or more, about 180 μm or more, about 185 μm or more, about 190 μm or more, about 195 μm or more, and the like.

In the present invention, a sample containing a certain amount of a nucleic acid-containing biomaterial can be provided by using a carrier capable of binding a nucleic acid-containing biomaterial together with a substrate having a lower binding ability for the nucleic acid-containing biomaterial than the carrier. It became easier, and it was possible to secure stability within several times between samples, and re-experimentation, which was necessary in the past, was substantially eliminated. In addition, detection of copy number variation was achieved with high reliability by using the present invention. In recent years, it has been clarified that gene regions with different copy numbers between individuals exist throughout the genome, and that they are responsible for diverse phenotypes. This is called copy number polymorphism, and it has been found that it is greatly involved in individual differences in drug metabolism, such as the observation of very complex CNPs in genes responsible for the metabolic system such as CYP2D6. there is In order to detect copy number polymorphism conventionally, Nested Long PCR or the like is complicated, requires a long time, and requires precise experimental conditions for diagnosis. Ta. There is no method for genetic analysis without DNA purification.

Here, by using a carrier capable of binding a nucleic acid-containing biomaterial and a substrate having a lower binding ability for the nucleic acid-containing biomaterial than the carrier, for example, a flat substrate having a certain area (e.g., swab-like) is used. The bound nucleic acid-containing biomaterial can be transferred from the substrate to a carrier capable of binding the nucleic acid-containing biomaterial in a range of amounts. Alternatively, a device designed to accommodate a certain volume (e.g., an applicator (Trust Catcher (registered trademark))) can be used to transfer nucleic acid-containing biomaterials to a carrier capable of binding in a certain range, Even in this case, it is preferable that the surface area is constant. In addition, since the volume effect (the sample absorbed inside seeps out) may exceed the detection limit, it is preferable that the volume portion is as small as possible. Alternatively, after the nucleic acid-containing biomaterial is supported on a base material having lower binding ability for the nucleic acid-containing biomaterial than the carrier, a certain area is cut out, and then transferred to a carrier capable of binding the nucleic acid-containing biomaterial. Can be transferred in a range of amounts. In another embodiment, after the nucleic acid-containing biomaterial is supported on a substrate having lower binding ability for the nucleic acid-containing biomaterial than the carrier, the nucleic acid-containing biomaterial is transferred to the carrier capable of binding, and then the carrier is subjected to a certain range. By cutting out an area (eg, using a biopsy punch or the like), a range of amounts of nucleic acid-containing biomaterial can be prepared. The technique of configuring such a "constant range amount" is exemplified in other places of this specification together with examples, and various implementations are possible.

In the present invention, PCR (Polymerase Chain Reaction) method and LAMP (Loop-Mediated Isothermal Amplification) method are carried out in the reaction solution after contact with the water-disintegrable carrier carrying the test sample. , SDA (Strand Displacement Amplification) method, RT-SDA (Reverse Transcription Strand Displacement Amplification) method, RT-PCR (Reverse Transcription Polymerase Chain Reaction) method, RT -LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, TMA (Transcription Mediated Amplification) method, RCA (Rolling Cycle Amplification) method, ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method, UCAN method, LCR (Ligase Chain Reaction) method, LDR (Ligase Detection Reaction: Ligase detection reaction) method, SMAP (Smart Amplification Process) method, SMAP2 (Smart Amplification Process Version 2) method, PCR-Invader method, Multiplex PCR-Based Real-Time Invader Assay (mPCR-RETINA) It is preferable to apply a method selected from These gene amplification methods are known per se.

As the DNA polymerase used in the present invention, any polymerase having excellent heat resistance for synthesizing a nucleic acid by addition of a primer, such as Taq DNA polymerase, can be used without particular limitation. Examples of such DNA polymerases include Taq DNA polymerase and Tth DNA polymerase derived from Thermus aquaticus, KOD DNA polymerase derived from Pyrococcus, Pfu or Pwo DNA polymerase, or a mixture of at least one of the above DNA polymerases. Among them, KOD DNA polymerase is preferred. In addition, Tth DNA polymerase and Carboxydothermus hydrogenoformans-derived C. Since the therm DNA polymerase also has RT activity, it has the characteristic that one kind of enzyme can be used when RT-PCR is performed in one tube-one step.

The buffer is not particularly limited as long as DNA can be amplified even in the presence of a substance that inhibits the PCR reaction contained in the test sample. Shimadzu Corporation), Phusion (registered trademark) Blood Direct PCR kit buffer (New ENGLAND Bio-Labs), KOD FX Neo buffer (manufactured by Toyobo Co., Ltd.), MasterAmp (registered trademark) PCR kit (manufactured by Epicentre), etc. It is particularly preferable to use KOD FX buffer (manufactured by Toyobo Co., Ltd.) developed for KOD DNA polymerase. For example, the PCR enzyme kit KOD FX Neo (manufactured by Toyobo Co., Ltd.) used in the examples contains 1 to 2M betaine, which has the effect of removing the reaction inhibition of PCR inhibitory components in the sample. MasterAmp (registered trademark) PCR kit (manufactured by Epicentre) states that it contains betaine, and is expected to have an effect equivalent to that of KOD FX Neo buffer.

Hereinafter, specific description will be given by taking as an example the case of performing the RT-PCR method, which is used as a suitable gene amplification method when the gene to be amplified or the specific gene to be detected is RNA.

The reaction solution (RT reaction solution) used for the reverse transcription (RT) reaction is usually pH buffer, salts such as MgCl ₂ and KCl, dithiothretol (DTT), primers, deoxyribonucleotides, RNase inhibitors, reverse transcriptase. includes. In addition, the above salts are appropriately changed to other salts before use. The RT reaction solution also usually contains proteins such as gelatin, albumin, and surfactants.

In the present invention, when the gene to be amplified or the specific gene to be detected is RNA, it is more preferable that the reaction solution containing the buffer and the DNA polymerase further contains a melting temperature adjusting agent. The melting temperature adjuster is for adjusting the melting temperature (Tm) between the primer and the template DNA during the DNA polymerase reaction. Examples of such melting temperature adjusters include betaine (N,N,N-trimethylglycine) Proline, dimethylsulfoxide (hereinafter abbreviated as DMSO), formamide, tetraalkylammonium salts and the like are generally used. By using a melting temperature adjusting agent, the annealing of oligonucleotides can be adjusted under limited temperature conditions. Furthermore, when betaine or a tetraalkylammonium salt is used as a melting temperature adjusting agent, it is effective in improving the efficiency of chain substitution due to its isstabilizing action.

When betaine, for example, is used as the melting temperature adjusting agent, its addition to the reaction solution in the range of 0.2 to 3.0 M, preferably 0.5 to 1.5 M can be expected to promote the nucleic acid amplification reaction according to the present invention. . Since these melting temperature modifiers act to lower the melting temperature, it is possible to appropriately set conditions that provide appropriate stringency and reactivity, taking into consideration other reaction conditions such as salt concentration and reaction temperature. can.

A reverse transcriptase used in the RT reaction refers to an enzyme that can reverse transcribe RNA into cDNA. Reverse transcriptases include reverse transcriptases from avian retroviruses such as Rous associated virus (RAV) and Avian myeloblastosis virus (AMV), reverse transcriptases from mouse retroviruses (MMLV) such as Moloney murine leukemia virus (MMLV). transcriptase, Tth DNA polymerase from Thermus thermophilus or C. Therm DNA polymerase and the like, but are not limited to these.

RT-PCR is performed by adding a part of the RT reaction product to the PCR reaction solution (Two tube-Two step), and by adding the PCR reaction solution to the RT reaction product (One tube-Two step step), or by preparing all the reagents necessary for RT-PCR in advance and performing the RT reaction and PCR continuously (One tube-One step).

In RT-PCR, the PCR reaction solution that follows the RT reaction usually contains a pH buffer, salts such as MgCl ₂ and KCl, primers, and deoxyribonucleotides. In addition, the above salts are appropriately changed to other salts before use. In addition, proteins such as gelatin and albumin, dimethylsulfoxide, surfactants, and the aforementioned melting temperature adjusters such as dimethylsulfoxide and betaine may also be added.

In RT-PCR, as an additive, for example, a solution containing 2.7 M betaine, 6.7 mM DTT, 6.7% DMSO, and 55 μg/mL BSA in 1 mL is prepared, and the concentration is adjusted and added as appropriate. You may do so. The same effect can be obtained by adding the above-mentioned commercially available MasterAmp (registered trademark) Enhancer (registered trademark) (with betaine) (ME81201, manufactured by Epicentre).

The primers used in the reaction solution of the present invention can be appropriately designed by known methods once the target gene to be amplified or the specific gene to be detected is determined. The primers used in the reaction solution of the present invention are not particularly limited as long as they are capable of specifically amplifying a gene to be amplified or a specific gene to be detected.

Amplification of a gene contained in a test sample or detection of a specific gene contained in a test sample in the present invention is preferably carried out on a plate-shaped or tube-shaped insoluble carrier. Examples of such an insoluble carrier include tubes made of plastic, glass, etc., which are insoluble in the reaction solution, and 96-well wells. Note that the tubular shape refers to a hollow state, and may be a shape such as a bottomed PCR tube or an Eppendorf tube.

Specifically, first, the reaction solution is put into a plate-shaped or tube-shaped insoluble carrier. In the case of a tubular insoluble carrier, a reaction solution containing reagents such as buffers, polymerases and primers is introduced into the carrier, and in the case of a plate-shaped insoluble carrier, the reaction solution is placed on the surface. . Then, the reaction solution, the test sample and the water-dissolvable carrier are arranged so as to be in direct contact, and the above-described PCR method, LAMP method, SDA method, RT-SDA method, RT-PCR method, RT-LAMP method, NASBA method, Select from TMA method, RCA method, ICAN method, UCAN method, LCR method, LDR method, SMAP method, SMAP2 method, PCR-Invader method, Multiplex PCR-Based Real-Time Invader Assay (mPCR-RETINA) apply the method Alternatively, the water-decomposable carrier supporting the sample to be tested may be placed on the insoluble carrier, and then the reaction solution may be placed on the insoluble carrier. FIG. 13 describes the operation after the substrate contains the nucleic acid-containing biomaterial. The nucleic acid-containing biomaterial is transferred from the substrate to the carrier, and a fixed area (4 mm diameter can be used here, but can be of any size, for example, 2-6 mm diameter, 3-5 mm diameter, or about 4 mm). diameter). This carrier is placed in a tube-shaped insoluble container with water for dissolution or the like, heated as necessary to elute the nucleic acid from the carrier or accelerate the disappearance of the carrier, and then a buffer, polymerase, primer and the like are added. An example of amplifying a gene or detecting a specific gene by adding an amplification reagent is shown (see FIG. 13 and FIG. 13A for a conventional example).

Detection can be performed by any technique in the art, such as TaqMan® probes, SYBR® Green, electrophoresis, DNA sequencing, and the like. These can be used to detect SNPs (single nucleotide polymorphisms), STRs (short tandem repeat polymorphisms, also called microsatellite polymorphisms), CNPs (copy number polymorphisms), and the like.

In the present invention, conditions for nucleic acid amplification (for example, PCR) are not particularly limited as long as specific amplification based on the principle of nucleic acid amplification occurs, and can be set as appropriate.

In one exemplary embodiment, the method for detecting a specific gene of the present invention includes contacting the above-described water-disintegrable carrier carrying the test sample with a reaction solution containing a buffer, a DNA polymerase and a primer, and performing an optical test. It also includes detecting whether or not a specific gene is contained in a reaction solution using a specific means. For example, a real-time PCR method can be used to detect a specific gene, and TaqMan (registered trademark) (Applied Biosystems) or the like, which is commonly used in the art, can be preferably used as an optical means used at that time. can. After amplification, in addition to detection of specific genes, for example, determination of copy number can also be performed.

When the real-time PCR method is employed, changes in fluorescence intensity are generally at the noise level and equal to zero for amplification cycles 1 to 10, so they are regarded as sample blanks with no amplification products, and their standard deviation SD is calculated. The fluorescence value multiplied by 10 is defined as a threshold value, and the PCR cycle number that first exceeds the threshold value is called the threshold cycle number (Ct value). Therefore, the larger the initial DNA template amount in the PCR reaction solution, the smaller the Ct value, and the smaller the template DNA amount, the larger the Ct value. Also, even if the amount of template DNA is the same, the greater the proportion of cleavage occurring in the specific gene of PCR within the template, the greater the Ct value of the PCR reaction in the same region.

The gene amplified from the test sample by the gene amplification method of the present invention may of course be subjected to detection or quantification. Alternatively, hybridization with beads, extension reaction using probe DNA, gene detection by hybridization, etc. can be mentioned, and detection using optical means may of course be used.

The present invention can also be applied to copy number variation. Application examples of copy number variation are described below.

Also called SNPs (single nucleotide polymorphisms, commonly referred to as single nucleotide polymorphisms), as is known in the art, they occur in >1% of the population. A polymorphism that literally differs by one nucleotide (single base). A repeat is a repetition of several bases, and repetitions of several times to several hundred times are found. A copy number variation (CNV) is a variation in genomic copy number, usually referring to sizes >1000 base pairs. In general, humans inherit two sets of genes, one from each parent's genome. Therefore, when focusing on a certain gene, it has been generally thought that each gene has two (=2 copies). However, in recent years, it has been found that there are individual differences in gene copy numbers (copy number polymorphism), such as the presence of only one copy of a certain gene or three or more copies of a gene per cell, depending on the individual. This copy number polymorphism is attracting attention as a cause of individual differences in constitution, such as differences in efficacy and side effects of various drugs. In previous studies, "differences in base sequences" of genes between individuals, typified by single nucleotide polymorphisms (SNPs), were well known. On the other hand, this phenomenon called copy number polymorphism is a "difference in number" of genes. Under this phenomenon, the sequence that contains the entire gene on the genome is duplicated or deleted, so the number of large regions, sometimes several thousand base pairs to several million base pairs, differs between individuals (http: //www.jst.go.jp/pr/info/info361/zu1.html). A variety of families can be targeted for copy number analysis, and there are normal families, families with low copy numbers, and families with high copy numbers. etc.) can be determined.

A method for detecting copy number variation is outlined. Commonly used CNV detection methods include methods targeting specific loci such as quantitative PCR and methods targeting the entire genome such as arrays. For example, array CGH is known, and this method, Comparative Genomic Hybridization (CGH), is an efficient method for examining DNA copy number changes for the entire genome (Fig. 6B ). In addition, in the SNP array, sample DNA is fragmented, labeled with a fluorescent dye, and reacted with the chip under heat denaturation conditions (Fig. 6C). A quantitative PCR method (Quantitative real time polymerase chain reaction: qPCR) can also be used. This is one of the most commonly used methods to screen for CNVs in targeted genomic regions. A primer is designed for the target gene (region), amplified by PCR while incorporating a fluorescent dye, and the amplification efficiency of the PCR product is measured. It is possible to quantify DNA (copy number analysis) by utilizing the difference in amplification efficiency depending on the amount of template (template DNA).

A method for detecting copy number variation using the TaqMan (registered trademark) method will be described. Compare the test sample (♂) with the calibration sample (♀). Test assays (for example YIPF6, ChrX, FAM) and reference assays (RNaseP, Chr14, for VIC) are performed and Ct values (number of cycles to reach a certain fluorescence intensity) are calculated. Calculate the Ct for the test and reference assays in the test sample. For example, if Ct(FAM) is 28.5 and Ct(VIC) is 27.0, the difference is ΔCt=1.5. When the calibration sample is similarly measured and Ct(FAM)=27.0 and Ct(VIC)=27.5 are calculated, ΔCt=0.5. Taking the difference ΔCt of these samples yields 1. Applying this value to 2 ^−ΔCt ×2 gives a copy number of 1 (see FIG. 6).

In addition to this, any known method can be used as a method for calculating copy number polymorphism. A step of typing the SNP site by the invader method using a DNA-containing sample containing the genomic region derived from the subject as a template, and measuring fluorescence in real time in the step. and how. A method is described for determining the copy number ratio of both alleles using the ratio of fluorescence intensities corresponding to each allele at a time point before the fluorescence intensity reaches saturation. Yijing He, et al., Trends Mol Med. 2011
May ; 17(5): 244-251 describes applications in pharmacogenetics. Whitney E. Kramer et al., Pharmacogenet Genomics. 2009 October; 19(10): 813-822 discusses copy number variations in CYP2D6 and their effects on metabolism and pharmacological action. . Japanese Patent Application Laid-Open No. 2008-49668 discloses a method for determining cancer predisposition using copy number variation. Philip S. Bernard et al., Clinical Chemistry 48:8 1178-1185 (2002) discuss the use of real-time PCT technology in cancer diagnosis and also copy number variation therein. Recently, Japanese Patent Laid-Open No. 2015-73506 also introduces a method of applying copy number polymorphism in the determination of genetic polymorphism. Thus, in a variety of ways, copy number variation can be performed and samples prepared using the present invention can be utilized.

By using the method for amplifying genes and the method for detecting specific genes of the present invention, it is possible to detect single nucleotide polymorphisms (SNPs) on the 3 billion base pairs human genome gene sequence. In addition to examining the genetic background from type determination, it is also possible to diagnose the future risk of genetic diseases for which the causative gene is known. For example, genetic factors such as resistance to alcohol can be examined by SNP typing the alcohol dehydrogenase gene (ADH1B) and the aldehyde dehydrogenase gene (ALDH2).

In the ADH1B polymorphism, the 47th arginine ( CGC ) is converted to histidine ( CAC ) (G of the second nucleic acid in the codon is converted to A), and guanine (G) is added to the SNP site. Those with ADH1B*1 allele and those with adenine (A) mutation are called ADH1B*2 alleles. Patients with the mutant ADH1B*2 allele, which is frequently found in Asia, have increased alcohol sensitivity due to an increased rate of acetaldehyde production due to increased ADH activity.

The ALDH2 gene polymorphism has 487th glutamic acid ( GAG ) converted to lysine ( AAG ) (G in the first nucleic acid in the codon is converted to A), and has guanine (G) at the SNP site. The ALDH2*1 allele is referred to as the ALDH2*1 allele, and the ALDH2*2 allele is mutated to have adenine (A). As for ALDH2, the mutant ALDH2*2 allele is frequently found in Asia, but the activity of ALDH2 is reduced in mutant carriers. It slows down the metabolism of acetaldehyde and increases alcohol sensitivity.

Suitable examples of genes to be genotyped include the gene for drug metabolizing enzyme CYP2C9 and the gene for vitamin K epoxide reductase VKORC1. Here, the drug-metabolizing enzyme CYP2C9 (Cytochtome P450, Family 2, Subfamily C, Polypeptide 9) has the function of metabolizing warfarin into a chemical structure that has lost efficacy in the body of the patient to whom it was administered. Here, about 96% of Japanese have normal CYP2C9 activity, but about 4% have low CYP2C9 activity, and about 1% have less than 1/10 enzyme activity. Known CYP2C9 genes include, for example, a gene CYP2C9*1 with normal enzymatic activity and a gene CYP2C9*3 with about 1/10 reduced enzymatic activity. That is, about 96% of Japanese with normal CYP2C9 activity have the allele CYP2C9 *1/*1, and about 4% of Japanese with low CYP2C9 activity have the allele CYP2C9 *1/*3. However, about 1% of Japanese whose enzyme activity is less than 1/10 have the allele CYP2C9 *3/*3.

Also, vitamin K epoxide reductase VKORC1 (Vitamin K Epoxide Reductase Complex Subunit 1) is an enzyme associated with blood coagulation. Here, vitamin K becomes reduced vitamin K, and this reduced vitamin K acts on the blood coagulation factor to activate the blood coagulation factor. Reduced vitamin K after acting on blood coagulation factors becomes vitamin K epoxide, and VKORC1 acts on this to reduce vitamin K epoxide to vitamin K. Warfarin inhibits the action of VKORC1, the rate-limiting enzyme in the vitamin K cycle, thereby preventing the formation of vitamin K from vitamin K epoxide and exerting an anticoagulant effect. This VKORC1 is also metabolized differently depending on the individual. As the VKORC1 gene, for example, the gene VKORC1 A/A and the gene VKORC1 G/A, which tends to increase the maintenance dose of warfarin, are known.

Other suitable examples of genes to be genotyped include β2 adrenergic receptor (ADRB2: Adrenergic, beta-2, receptor, surface) gene, β3 adrenergic receptor (ADRB3: Adrenergic, beta-3 , receptor, surface) gene, uncoupling protein 1 (UCP1: Uncoupling Protein 1) gene, and the like. It is known that each of these genes also has a genetic polymorphism, and that the basal metabolism (resting calorie consumption) differs depending on the genotype. Here, ADRB2 is mainly distributed in the heart, bronchial smooth muscle, prostate, etc., and is also present in adipose tissue and participates in lipolysis. ADRB3 is found primarily in adipose tissue and is involved in regulating lipolysis and thermogenesis. In addition, UCP1 is specifically expressed in brown adipocytes and is involved in thermogenesis during cold weather and after meals.

In addition, it is a protein that is expressed only in fast-twitch fibers, and is a so-called "sports gene" that provides clues for determining whether the difference in genotypes may have aptitude for exercise that requires explosive power or endurance. The ATCN3 (Alpha-Actinin-3) gene, which has been attracting attention, may be used as a genotype determination target, and ABCC11 (ATP-binding cassette transporter sub -Family C member 11) genes and EDAR (Ectodysplasin A Receptor) genes, which are known to be involved in individual and group differences in facial morphology, may be used as targets for genotype determination.

In addition to the above-mentioned single nucleotide polymorphism (SNP) for diagnosing a person's constitution, the method for amplifying a gene and the method for detecting a specific gene of the present invention can also It can also be applied to microbiological examinations such as.

In recent years, it has been clarified that gene regions with different copy numbers among individuals exist throughout the genome, and that they are responsible for various phenotypes. This is called copy number polymorphism, and a very complex CNP is also observed in the CYP2D6 gene used in our study, which is greatly involved in individual differences in drug metabolism. The present inventors developed a nested long PCR method using dried saliva specimens for polymorphism analysis of the gene-deficient CYP2D6 ^* 5 gene, but the reaction required a long time and it was difficult to process multiple specimens. there were. Diagnosis of copy number variation requires preparation of precise experimental conditions, and there is absolutely no method for gene analysis without DNA purification. As with single nucleotide polymorphisms, stabilization of the starting material was an important factor for the detection of CYP2D6 ^* 5 (deficient) copy number polymorphisms using dried saliva. The present invention develops a technique for providing nucleic acid-containing biological samples such as cells within a certain range, thereby suppressing this "certain range" to a range in which non-specific amplification does not affect copy number. In addition to polymorphisms, we have perfected the technology to detect and sequence SNPs. In particular, the surface area of the "substrate having a lower ability to bind nucleic acid-containing biomaterials than the carrier" used in the present invention is characterized in that it is sufficient to provide the aforementioned range of amounts, in another embodiment , being equal to or wider than the fixed area, and from the problem of operability, the substrate is characterized by a shape sufficient to cover the fixed area, thereby contributing to a simple rapid analysis method. preparation was achieved. Further, in an embodiment using a carrier that allows optical detection, the configuration of the present invention can be used for a simple and rapid analysis method that is suitable even when nucleic acids in the sample after nucleic acid amplification are optically detected. Favorable sample preparation was achieved.

(preferred embodiment)
Preferred embodiments of the present invention are described below. The embodiments provided below are provided for a better understanding of the invention, and it is understood that the scope of the invention should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present invention in light of the description in this specification. It is also understood that the following embodiments of the invention can be used singly or in combination.

<Sample preparation for nucleic acid amplification>
In one aspect, the present invention includes a nucleic acid-containing biomaterial comprising (A) a carrier capable of binding a nucleic acid-containing biomaterial and (B) a substrate having a lower affinity for binding the nucleic acid-containing biomaterial than the carrier. A kit for preparing a sample for nucleic acid amplification from a sample (also referred to herein as a "sample preparation kit") is provided. The present invention requires two types of materials, and has the property that it has the ability to bind nucleic acid-containing biomaterials (e.g., cells) in the order of (A carrier)>(B base material). The inventors have found that a sample for nucleic acid amplification can be prepared from a sample containing a large amount of nucleic acid-containing biomaterials. These carriers and substrates can be identified by the determination methods described herein. Substrate (B) is configured to provide a sample containing a range of amounts of nucleic acid-containing biomaterial. Such a configuration may, for example, be configured in such a way that a fixed area can be contacted with the carrier, or configured to excise a fixed area before or after contact. Carrier (A) preferably allows nucleic acid amplification when placed in water. This is because the nucleic acid amplification reaction can be started immediately after sample preparation. In a preferred embodiment, carrier (A) advantageously allows nucleic acid amplification when placed in water. This is because the carrier (A) is bound with nucleic acid-containing biomaterials (eg, cells), and by using this as it is, the time until nucleic acid amplification can be shortened. Furthermore, it is possible to omit complicated steps such as washing of the substrate/carrier, thereby making it possible to simplify the process, and in addition, it is possible to prevent cross-contamination accompanying washing and the like.

In one preferred embodiment, the sample preparation kit of the present invention comprises: (A) a hydrolyzable carrier (preferably allowing optical detection); and a substrate that provides a sample comprising. A range amount, as used herein, is an amount that allows for genetic testing after nucleic acid amplification. For example, an amount that allows discriminating copy number after nucleic acid amplification. Examples of such amounts are within X times to 1/X with respect to a certain reference amount, and X is an amplification factor that can be distinguished when the copy number differs by 1 in the nucleic acid amplification (e.g., 6 cycles In this case, X is 64, 5 cycles In this case, X is 32, 4 cycles In this case, X is 16, 3 cycles In this case, X is 8, 2 cycles In this case, X is 4, 1 cycles In this case, X is 2) is the difference. Alternatively, in the measurement or analysis performed after nucleic acid amplification in the present invention, it is an amount that avoids confusion with another result, and is within 64 times to 1/64 of the reference amount, and X ² is the difference in the amplification factor when the copy number differs by 1 in the nucleic acid amplification, for example, the square root of 10 to 1, 3 to 1/3, 1.7 to 1.8 About 1/1.7 to 1/1.8, etc. can be mentioned. Alternatively, it is within X times to 1/X with respect to the reference amount, where X is a identifiable fold difference when the copy number differs by 1 in the nucleic acid amplification.

INDUSTRIAL APPLICABILITY The present invention makes it possible to realize an ultra-simple personal genome information analysis apparatus that is fully automated, from sampling to genetic analysis, without requiring an operator trained in genetic testing. The present invention provides a genetic testing technique for detecting not only single nucleotide polymorphisms (SNPs) but also microsatellite polymorphisms (STRPs) and copy number polymorphisms (CNPs) directly and simultaneously in multiple items with high sensitivity and ultra-high speed. According to the present invention, it is also possible to prepare a reaction solution corresponding to multi-specimen, multi-item genetic testing. For example, in combination with high-speed PCR technology, sample preparation to analysis including PCR technology can be performed on demand within 15 minutes. It also makes it possible to complete

Specifically, the present invention provides the most efficient method of sampling cells, such as oral mucosal cells. The present invention optimizes the transfer and rapid drying method using the carrier developed by the present inventors and provides advantages in the structure, shape and operability of the sampling device. The present invention can be applied to various automatic reaction liquid adjustment devices. Since the present invention has a structure that can hold the maximum amount of cells such as oral mucosal cells per unit area of the water-dissolvable carrier, copy number variation (CNV) or gene expression profiling for quantifying the copy number of genes can be performed very easily. can be advantageously implemented in

Advantageously, the carrier used in the present invention may be any carrier that does not terminate nucleic acid amplification, and preferably allows optical detection. . It is understood that even those that stop or inhibit nucleic acid amplification can be used in the present invention as long as they can be removed or reduced during the amplification reaction. One embodiment of the water-disintegratable carrier described herein provides these properties, and various embodiments can be used as described herein.

One preferred embodiment is characterized in that the surface area of the substrate (B) used in the present invention is sufficient to provide a range of amounts of nucleic acid-containing biomaterial. Without wishing to be bound by theory, when a sample is collected in a fixed volume, as typified by a dropper, the level of quantification of the finally obtained sample may not be as expected. In some cases it was necessary to repeat the assay again with more than 10% of the sample, but quantification could be further improved if it was ensured that it was sufficient to provide a range of amounts on a surface area basis. shown. Without wishing to be bound by theory, the reason why quantification is ensured by keeping the surface area constant is that the contact surface is important for keeping nucleic acid-containing biomaterials such as cells constant. Therefore, the configuration of the present invention can significantly reduce the need for retesting. In another embodiment, the substrate (B) used in the present invention may have a surface area equal to or greater than a defined area that provides a range of amounts of nucleic acid-containing biomaterial. More preferably, in consideration of the problem of operability, etc., the base material (B) used in the present invention has a shape sufficient to cover the above-mentioned fixed area (when securing a fixed area by cutting, a shape to be cut can be.) can be. This is because if the shape is different from that of the base material or the shape to be excised, there is a possibility that the nucleic acid-containing biomaterial transferred will not be a constant amount even if it has a constant area. In this way, by adopting a configuration in which a constant area is secured for the surface to which the nucleic acid-containing biomaterial is transferred, sample preparation that contributes to a simple rapid analysis method while improving quantification has been achieved. This also solved the problems associated with materials prepared on a volume basis.

Further, in an embodiment using a carrier that allows optical detection (for example, preferably hydrolytic paper), by using the configuration of the present invention, when the nucleic acid in the sample after nucleic acid amplification is optically detected The sample preparation contributing to a suitable simple rapid analysis method was also achieved.

Therefore, in one specific embodiment, examples of carriers used in the present invention include water-disintegrable carriers (e.g., water-disintegrable paper), Kimwipes, Kimtowels, newspapers, filter papers, and the like. Preferably, it is a water-disintegrable carrier (eg, water-disintegrable paper). On the other hand, when using newspaper or the like, it may be advantageous to perform a treatment such as heating after contact.

The "substrate" used in the present invention preferably provides a sample containing a range of nucleic acid-containing biomaterials. In this embodiment, when the purpose is to provide a sample containing a nucleic acid-containing biomaterial in a certain amount, a sample containing the nucleic acid-containing biomaterial (for example, saliva containing oral mucosal cells, etc.) can be retained. Any material can be used as long as it is a material. Advantageously, such substrates are substrates that carry nucleic acid-containing biomaterials (eg, cells, tissues, bacteria, viruses, samples prepared therefrom, etc.) without binding. In particular, if at least the nucleic acid in these nucleic acid-containing biomaterials can be carried without binding, it can function for the purpose of the present invention, so it can be determined that the nucleic acid-containing biomaterial is carried without binding. . Examples of such substrates include polyurethane, polyvinyl alcohol (PVA), polyethylene, chitin, chitosan, collagen, polyvinylidene difluoride (PVDF), polypropylene, cellulose and its derivatives, paper, collagen, gelatin, urethane. , silicone, absorbent cotton (cellulose absorbent cotton, acetate absorbent cotton), combinations thereof, or mixtures thereof. Polyurethane, polyvinyl alcohol (PVA), and polyethylene are preferably used. These materials are capable of holding samples containing nucleic acid-containing biomaterials. Examples of such materials include, but are not limited to, those described in Patent Document 2, International Publication No. 2011/016423, and Utility Model No. 3169659.

In the present specification, methods for determining whether or not the base material of the present invention has the function of supporting a nucleic acid-containing biomaterial without binding include binding capacity comparison method (1), binding capacity comparison method (2), and the like. be able to. Whether or not the nucleic acid-containing biomaterial is supported without binding may be a relative relationship with other carriers that are contacted.

Examples of such substrates include swabs (e.g., Isohelix DNA Buccal Swabs (Isohelix)), cards (Whatman® FTA® cards (GE Healthcare)), Trust Catcher® (registered trademark). Utility model: see No. 3169659) liquid sample collection kit, dropper, etc., but not limited to these.

Typically, the carriers and substrates that can be employed in the sample preparation kits of the present invention contain a sufficient number of cells ( calculated to release approximately 1×10 ¹ copy cells) can be utilized. Substrates or materials that are "releasable under heating conditions" or "releasable under boiling conditions" and "capable of binding nucleic acid-containing biomaterials" may be used. In addition, the base material having a lower binding ability for nucleic acid-containing biomaterials than the carrier of the present invention is selected from the materials selected in the test of (Nucleic acid-containing biomaterial binding test) by binding ability comparison method (1) or (binding ability comparison method Any material determined by (2) or the like to have lower affinity for nucleic acid-containing biomaterials than the carrier employed in the present invention can be used.

Combinations of such substrate and carrier materials include the following, all of which are included in preferred embodiments of the present invention.
(Base material: carrier) = polyurethane (swab): water-dissolvable paper (for example, 30MDP, 60MDP, 120MDP, 30CD, 60CD, 120CD (manufactured by Nippon Paper Papylia Co., Ltd.))
(Base material: carrier) = polyvinyl alcohol (PVA; trust catcher): water-dissolvable paper (e.g., 30MDP, 60MDP, 120MDP, 30CD, 60CD, 120CD (manufactured by Nippon Paper Papylia Co., Ltd.))
(Base material: Carrier) = Absorbent cotton (cotton swab): Water-dissolving paper (e.g., 30MDP, 60MDP, 120MDP, 30CD, 60CD, 120CD (manufactured by Nippon Paper Papylia Co., Ltd.))
(Base material: carrier) = polyester (cotton swab): water-dissolving paper (e.g., 30MDP, 60MDP, 120MDP, 30CD, 60CD, 120CD (manufactured by Nippon Paper Papylia))
(Base material: carrier) = urethane sponge (sponge swab): water-dissolvable paper (e.g., 30MDP, 60MDP, 120MDP, 30CD, 60CD, 120CD (manufactured by Nippon Paper Papylia Co., Ltd.))
(Base material: carrier) = natural sponge: water-dissolvable paper (for example, 30MDP, 60MDP, 120MDP, 30CD, 60CD, 120CD (manufactured by Nippon Paper Papylia Co., Ltd.))
(Base material: carrier) = polyurethane (swab): qualitative filter paper (for example, AD-1, AD-2, AD-3, AD-4A, AD-6 (manufactured by Advantech))
(Base material: carrier) = polyvinyl alcohol, PVA (trust catcher): qualitative filter paper (for example, AD-1, AD-2, AD-3, AD-4A, AD-6 (manufactured by Advantech))
(Base material: carrier) = absorbent cotton (cotton swab): qualitative filter paper (for example, AD-1, AD-2, AD-3, AD-4A, AD-6 (manufactured by Advantech))
(Base material: carrier) = polyester (cotton swab): qualitative filter paper (for example, AD-1, AD-2, AD-3, AD-4A, AD-6 (manufactured by Advantech))
(Base material: carrier) = urethane sponge (sponge swab): qualitative filter paper (for example, AD-1, AD-2, AD-3, AD-4A, AD-6 (manufactured by Advantech))
(Substrate: carrier) = natural sponge: qualitative filter paper (eg, AD-1, AD-2, AD-3, AD-4A, AD-6 (manufactured by Advantech)).

When swabs are used, the range of doses defined in the present invention is achieved by removing the water-disintegratable carrier in a constant area. Extraction in a certain area can be achieved using an instrument or the like capable of cutting the water-disintegrable carrier, for example, an excision device capable of excising a certain area (e.g., Biopsy Trepan (Kai), Harris Micropunch (GE Healthcare), Harris Unicore Punch (GE Healthcare), etc. Available swabs may be home made, GE Healthcare (Sterile Omni Swab) , Sterile Foam Tipped Applicator, etc.), Texwipe (Circular Head Swab TX708A, etc.).

Even when a liquid sample collection kit (for example, Trust Catcher (registered trademark)) is used, quantification is ensured by contact. It can also be achieved by providing

The present invention provides a technique for stably providing nucleic acid-containing biomaterials (eg, cells) in a certain amount. In Patent Document 2, etc., a method was developed based on a method in which a medium obtained by applying saliva to water-soluble paper and drying it is directly subjected to a real-time PCR reaction. However, the number of oral mucosa cells in saliva varies, and it was found that about 10% of the samples required re-experimentation. The present invention focuses on using a material that can directly adsorb oral mucosa cells on the inside of the cheek instead of saliva and that can transfer the cells to water-soluble paper. Not only is it dramatically improved, but when the water-soluble paper is punched out and added to the PCR reaction solution, the number of cells (DNA amount) existing on the water-soluble paper is kept within several times between samples. By doing so, a technique is provided that enables easy identification of copy number variation. The present invention develops a technique for providing nucleic acid-containing biological samples such as cells within a certain range, thereby suppressing this "certain range" to a range in which non-specific amplification does not affect copy number. A technique for detecting polymorphism is provided. Using the nucleic acid sample provided by the present invention, copy number variation can be assayed easily. Here, if necessary, the experimental conditions for stabilizing the PCR reaction may be examined, the internal standard may be appropriately selected, and the evaluation method may be appropriately prepared.

There are various methods for copy number polymorphism analysis, and when targeting genes with known copy number polymorphisms, conventionally, long template PCR and comprehensive analysis of copy number polymorphism at the chromosomal level are used. In addition to microarrays, FISH, Southern blotting, and the like have been used. CGH microarrays are ideal in terms of speed and convenience, and improvements in technology have enabled analysis at the level of almost each gene, and are often used in copy number variation analysis methods. However, it requires high technical skills, has problems in terms of cost, and has problems in processing the enormous amount of data that can be comprehensively obtained. The real-time PCR method, which is assumed to use the present invention, is simple and excellent in quantification, and can accurately measure the copy number. According to the present invention, in the prior art, it was necessary to accurately quantify template DNA obtained by extracting and purifying DNA from biological samples before performing genetic testing. Copy number variation genetic testing is possible without sacrificing quality, and there is a huge advantage in that skilled operators and technicians are not required for the operation associated with genetic testing, and it is overwhelmingly cheap and fast.・A non-invasive, simple, highly reliable and accurate genetic polymorphism analysis system is provided. In relation to copy number polymorphism, it is expected that copy number polymorphism in cancer is related to anticancer drug sensitivity, and genetic diagnosis of drug sensitivity and disease-related genes will become immediately possible in clinical settings. Various applications are expected, such as improvement of drug treatment rate and reduction of side effects. Immediate diagnosis is a powerful tool for elucidating interactions between drugs, foods, supplements, etc., in urgent treatment policy decisions such as drug eruptions, drug allergies, and drug addiction symptoms.

In one preferred embodiment, the carrier capable of binding nucleic acid-containing biomaterials used in the present invention allows nucleic acid amplification and optical inspection even when present in water, which is the object of the present invention. Any permissible material can be used in the present invention. The carrier used in the present invention is preferably a water-disintegratable carrier, and a representative water-disintegratable carrier is water-disintegratable paper. Such water-disintegratable carriers and water-disintegratable papers are also described in detail in Patent Document 2 (Japanese Patent No. 5875230).

In one embodiment, the nucleic acid amplification utilized in the present invention is a nucleic acid amplification method that results in non-specific amplification. In nucleic acid amplification methods that cause non-specific amplification, it is necessary to ensure specific amplification by limiting the amount of nucleic acid sample used as the starting material within a certain range in order to determine whether the amplification is specific. is important.

In one aspect, the present invention provides a kit for amplifying nucleic acid without purification (herein referred to as "nucleic acid amplification (also referred to as a “kit”). It is understood that the sample preparation kits of the present invention used herein can utilize one or more combinations of any of the embodiments described herein.

In one embodiment, reagents for nucleic acid amplification used in the nucleic acid amplification kit of the present invention may contain primers, nucleic acid amplification enzymes, and necessary buffers. These reagents may be provided in a kit or may be provided separately.

In one preferred embodiment, the carrier ((A) in the kit) capable of binding the nucleic acid-containing biomaterial of the present invention may be provided together with a non-water-absorbing material and/or a water-absorbing material. In such cases, the water-disintegratable carrier may take the form of a device for drying the biological sample, comprising (X) a non-water-absorbing material and (Y) a carrier capable of binding the nucleic acid-containing biomaterial. The device for drying the biological sample may further comprise (Z) a water absorbent material.

Therefore, in another aspect, the present invention provides a method for drying a biological sample comprising (X) a non-water-absorbing material, (Y) a carrier capable of binding a nucleic acid-containing biological material, and (Z) a water-absorbing material. (also referred to herein as a "biological sample drying device"). Any embodiment described herein can be adopted for the carrier capable of binding the nucleic acid-containing biomaterial used here.

In one embodiment, the biological sample drying device of the present invention or a carrier capable of binding a nucleic acid-containing biological material is arranged so that the non-water-absorbing material holds the carrier, and the non-water-absorbing material binds to the water-absorbing material. is detachably connected to the The non-water-absorbing material used in the kit, device, etc. of the present invention may be connected to the water-absorbing material via a removable adhesive member. In one embodiment, the non-water-absorbing material used in the kit, device, etc. of the present invention may be arranged to surround the water-disintegrable carrier.

In one embodiment, the carrier capable of binding the nucleic acid-containing biomaterial used in the kit, device, etc. of the present invention may be in contact with the non-water-absorbing material and the water-absorbing material, or may contact the water-absorbing material. It may be arranged so as not to be in direct contact with the material. By arranging the carrier so as not to come into direct contact with the water-absorbing material, it is possible to prevent the carrier capable of binding the nucleic acid-containing biomaterial from unintentionally coming into contact with water.

In one preferred embodiment, the carrier (Y) may allow nucleic acid amplification when placed in water. In another preferred embodiment, the carrier (Y) preferably allows optical detection. In a representative embodiment, carrier (Y) is a water-disintegratable carrier.

In another aspect, the present invention provides a method for detecting a specific gene contained in a nucleic acid-containing biomaterial and/or the copy number of the specific gene, comprising: a carrier capable of binding the nucleic acid-containing biomaterial; a step of providing a test sample containing the nucleic acid-containing biomaterial with a water-dissolvable carrier using a base material having a lower binding ability for the nucleic acid-containing biomaterial than the carrier; The step of contacting with an aqueous solution containing a nucleic acid amplification reagent containing a primer that enables the nucleic acid to be amplified, using the optical means to determine whether or not the specific gene is contained in the aqueous solution and/or the identification A method is provided comprising detecting the copy number of a gene. Here, by using this method, the nucleic acid-containing biomaterial is provided in an amount that allows identification after nucleic acid amplification (for example, an amount that allows identification of the copy number after nucleic acid amplification), so that highly reliable identification A method of detecting the copy number of a gene and/or said particular gene is achieved.

As used herein, the term "an amount that enables discrimination after nucleic acid amplification" refers to an amount that can be distinguished from non-specific amplification of other genes when a nucleic acid amplification reaction is performed. As used herein, the term "an amount capable of discriminating the copy number after nucleic acid amplification" refers to an amount capable of distinguishing from non-specific amplification of genes having other copy numbers. "Amount that allows identification after nucleic acid amplification" and "an amount that allows identification of copy number after nucleic acid amplification" may refer to substantially the same amount, and are also used herein as " It can also be an amount that is substantially the same as a "range amount". For example, within X times to 1/X of the reference amount, ^X2 is the difference in amplification factor when the copy number differs by 1 in the nucleic acid amplification.

As used herein, the term "primer that enables specific amplification of a specific gene" refers to a primer for nucleic acid amplification that is capable of amplifying a specific gene of interest more than other genes. .

In one embodiment, a test sample containing the nucleic acid-containing biomaterial is provided by contacting the substrate with the nucleic acid-containing biomaterial and then contacting the substrate with the carrier.

In another embodiment, the test sample is provided to contain a range of amounts of nucleic acid-containing biomaterials.

In one embodiment, the kit, device, etc. of the present invention can be applied to a method of detecting a specific gene contained in a test sample, which is blood or saliva containing intraoral cells. In this method, a water-disintegratable carrier carrying a test sample is brought into contact with a reaction solution containing a buffer, a DNA polymerase, and a primer, and the water-disintegratable carrier is decomposed in the reaction solution, thereby preventing the water-disintegratable carrier from interfering with the test sample. Alternatively, the method may be characterized by detecting whether or not the specific gene is contained in the reaction solution after decomposition of the water-decomposable carrier using CCD-type optical means. As optical means, UV (spectrophotometer), visible meter (turbidity meter), colorimeter (absorption photometer), fluorometer, etc. can be used in addition to the naked eye, CCD system, and PMT system.

One of the characteristics of the kit, device, etc. of the present invention is that the copy number can be finally detected only by optical detection (including discrimination between 0 and 1). Therefore, the kit of the present invention can be applied to kits containing parts other than optical means and amplification devices, characterized in that the substrate carrying a nucleic acid-containing biological sample (e.g., cells) has a "constant surface area." and may be applied to systems that include amplification devices in addition to this kit, and/or systems that include optical means.

In one aspect, the present invention provides a method for detecting a specific gene contained in a nucleic acid-containing biomaterial and/or the copy number of the specific gene, comprising: (A) a carrier capable of binding a nucleic acid-containing biomaterial; B) a step of providing a test sample containing the nucleic acid-containing biomaterial with a water-degradable carrier using a substrate having a lower binding ability for the nucleic acid-containing biomaterial than the carrier; a step of bringing into contact with an aqueous solution containing a nucleic acid amplification reagent containing primers capable of selective amplification and subjecting it to conditions for nucleic acid amplification, using the optical means to determine whether the specific gene is contained in the aqueous solution and/ Alternatively, a method is provided comprising the step of detecting the copy number of said specific gene. Here, by using (A) a carrier capable of binding a nucleic acid-containing biomaterial and (B) a substrate having a lower binding ability to the nucleic acid-containing biomaterial than the carrier, an amount that enables identification after amplification. Alternatively, an amount capable of distinguishing copy number after nucleic acid amplification is provided. Nucleic acid-containing biomaterials used as test samples include, for example, nasal secretions, nasal swabs, conjunctival swabs, pharynx swabs, sputum, feces, blood, serum, plasma, cerebrospinal fluid, saliva, urine, sweat, and milk. , semen, oral swabs, interdental swabs, wet cerumen, vaginal swabs, food and tissue, and the like.

In one embodiment, also in the method of the present invention, PCR method, LAMP method, SDA method, RT-SDA method, RT A reaction selected from -PCR method, RT-LAMP method, NASBA method, TMA method, RCA method, ICAN method, UCAN method, LCR method, LDR method, SMAP method, and SMAP2 method can be performed.

As the DNA polymerase that can be contained in the nucleic acid amplification reagent-containing aqueous solution of the present invention, any polymerase having excellent heat resistance that synthesizes nucleic acid by primer addition, such as Taq DNA polymerase, can be used without particular limitation. Examples of such DNA polymerases include Taq DNA polymerase and Tth DNA polymerase derived from Thermus aquaticus, KOD DNA polymerase derived from Pyrococcus, Pfu or Pwo DNA polymerase, or a mixture of at least one of the above DNA polymerases. Among them, KOD DNA polymerase is preferred. In addition, Tth DNA polymerase and Carboxydothermus hydrogenoformans-derived C. Since the therm DNA polymerase also has RT activity, it has the characteristic that one kind of enzyme can be used when RT-PCR is performed in one tube-one step as described later.

The buffer that can be contained in the nucleic acid amplification reagent-containing aqueous solution of the present invention is not particularly limited as long as it is capable of amplifying DNA even in the presence of a substance that inhibits the PCR reaction. ) (manufactured by Shimadzu Corporation), Phusion (registered trademark) Blood Direct PCR kit buffer (New ENGLAND Bio-Labs), KOD FX buffer (manufactured by Toyobo Co., Ltd.), MasterAmp (registered trademark) PCR kit (manufactured by Epicentre ) and the like are preferably used, and it is particularly preferable to use KOD FX buffer (manufactured by Toyobo Co., Ltd.) developed for KOD DNA polymerase. For example, the PCR enzyme kit KOD FX Neo (manufactured by Toyobo Co., Ltd.) used in the examples contains 1 to 2M betaine, which has the effect of removing the reaction inhibition of PCR inhibitory components in the sample. Note that the MasterAmp (registered trademark) PCR kit (manufactured by Epicentre) states that it contains betaine, and is expected to have an effect equivalent to that of the KOD FX buffer.

In another aspect, the present invention provides a kit for providing a sample for detecting a specific gene contained in a nucleic acid-containing biomaterial and/or the copy number of the specific gene using optical means, comprising (A) A kit for providing a test sample containing a nucleic acid-containing biomaterial, comprising a carrier capable of binding a nucleic acid-containing biomaterial, and (B) a substrate having a lower affinity for the nucleic acid-containing biomaterial than the carrier, Provided is a kit comprising a primer that enables specific amplification of a specific gene, and a nucleic acid amplification reagent or an aqueous solution containing the nucleic acid amplification reagent. The substrates, carriers, primers, nucleic acid amplification reagents, etc. used herein can utilize any of the embodiments described herein. Here, when (A) a carrier capable of binding a nucleic acid-containing biomaterial and (B) a substrate having a lower binding ability for the nucleic acid-containing biomaterial than the carrier is used, the nucleic acid-containing biomaterial such as cells is used. A substrate having a certain surface area that supports without binding and a carrier that binds the nucleic acid-containing biomaterial may be used.

In another aspect, the present invention provides a system for detecting a specific gene contained in a nucleic acid-containing biomaterial and/or the copy number of the specific gene, comprising: (A) a carrier capable of binding the nucleic acid-containing biomaterial; B) a kit for providing a test sample containing a nucleic acid-containing biomaterial, the kit comprising a substrate having a lower binding ability for the nucleic acid-containing biomaterial than the carrier, and primers enabling specific amplification of the specific gene. , a system comprising a nucleic acid amplification reagent or a nucleic acid amplification reagent-containing aqueous solution, a nucleic acid amplification device, and an optical means. (A) a carrier capable of binding a nucleic acid-containing biomaterial, and (B) a substrate having a lower affinity for binding the nucleic acid-containing biomaterial than the carrier, is a certain carrier that supports a nucleic acid-containing biomaterial such as cells without binding. It may be a substrate having a surface area and a water-degradable carrier that binds the nucleic acid-containing biomaterial. Substrates, carriers, primers, nucleic acid amplification reagents, optical means, etc. used herein can utilize any of the embodiments described herein.

Alternatively, the present invention is a system for detecting a specific gene contained in a nucleic acid-containing biomaterial, comprising a device comprising a non-water-absorbing material, a water-dissolvable carrier that binds the nucleic acid-containing biomaterial, and a water-absorbing resin, A kit for providing a test sample containing a nucleic acid-containing biomaterial, a primer that enables specific amplification of the specific gene, a nucleic acid amplification reagent or an aqueous solution containing the nucleic acid amplification reagent, a nucleic acid amplification device, and optical means. Provide a system. Substrates, carriers, primers, nucleic acid amplification reagents, optical means, etc. used herein can utilize any of the embodiments described herein. Examples of optical means include, but are not limited to, arbitrary analysis means for capturing an image with the naked eye, a CCD image sensor, a PMT (Photomultiplier Tube), a CMOS image sensor, or the like.

Another improvement provided by the present invention is the rapid drying feature of biological samples. Features corresponding to this part can also be used in combination with other detection method and apparatus parts of the invention.

The kit and device for realizing rapid drying of a biological sample of the present invention can be applied, for example, to a method for detecting a specific gene contained in a nucleic acid-containing biological material. Moreover, the kit, device, and the like for realizing rapid drying of the biological sample of the present invention may be used by being incorporated into a kit including parts other than the optical means/amplifier. Alternatively, the kit, device, etc. for realizing rapid drying of biological samples of the present invention may be applied to a system including an amplification device and/or optical means.

In one aspect, the present invention provides a method for detecting a specific gene contained in a nucleic acid-containing biomaterial, wherein a test sample containing the nucleic acid-containing biomaterial is combined with a non-water absorbent material and a nucleic acid-containing biomaterial. a step of contacting a device containing the obtained carrier and a water-absorbing resin, contacting the carrier with an aqueous solution containing a nucleic acid amplification reagent containing a primer that enables specific amplification of the specific gene, and subjecting the aqueous solution to conditions for nucleic acid amplification and detecting whether or not the specific gene is contained in the aqueous solution and/or the copy number of the specific gene using the optical means. Nucleic acid-containing biomaterials, non-water-absorbing materials, carriers, water-absorbing resins, primers, nucleic acid amplification reagents, optical means, etc. used here can be used in any of the embodiments described herein. is understood.

In another aspect, the present invention is a kit for providing a sample for detecting a specific gene contained in a nucleic acid-containing biomaterial, comprising a non-water-absorbing material, a carrier capable of binding the nucleic acid-containing biomaterial, and a water-absorbing resin. a kit for providing a test sample containing a nucleic acid-containing biomaterial, a primer that enables specific amplification of the specific gene, and a nucleic acid amplification reagent or an aqueous solution containing the nucleic acid amplification reagent, comprising: Provide a kit. Nucleic acid-containing biomaterials, non-water-absorbing materials, carriers, water-absorbing resins, primers, nucleic acid amplification reagents, optical means, etc. used here can be used in any of the embodiments described herein. is understood.

In another aspect, a system for providing a sample for detecting a specific gene contained in a nucleic acid-containing biomaterial, the device comprising a non-water-absorbing material, a carrier capable of binding the nucleic acid-containing biomaterial, and a water-absorbing resin. a kit for providing a test sample containing a nucleic acid-containing biomaterial, a primer that enables specific amplification of the specific gene, a nucleic acid amplification reagent or a nucleic acid amplification reagent-containing aqueous solution, and a nucleic acid amplification device, Provide a system. Nucleic acid-containing biomaterials, non-water-absorbing materials, carriers, water-absorbing resins, primers, nucleic acid amplification reagents, optical means, nucleic acid amplification devices, etc. used herein may be any of the embodiments described herein. can be used.

In another aspect, the present invention provides a system for detecting a specific gene contained in a nucleic acid-containing biomaterial, comprising a device comprising a non-water-absorbing material, a carrier capable of binding the nucleic acid-containing biomaterial, and a water-absorbing resin. a kit for providing a test sample containing a nucleic acid-containing biomaterial, a primer that enables specific amplification of the specific gene, a nucleic acid amplification reagent or an aqueous solution containing the nucleic acid amplification reagent, a nucleic acid amplification device, and an optical means and providing a system. Nucleic acid-containing biomaterials, non-water-absorbing materials, carriers, water-absorbing resins, primers, nucleic acid amplification reagents, optical means, nucleic acid amplification devices, etc. used herein may be any of the embodiments described herein. can be used.

When the real-time PCR method is employed, changes in fluorescence intensity are generally at the noise level and equal to zero for amplification cycles 1 to 10, so they are regarded as sample blanks with no amplification products, and their standard deviation SD is calculated. The fluorescence value multiplied by 10 is taken as a threshold value (threshold, limen), and the number of PCR cycles that first exceeds the threshold value is called the cycle threshold value (Ct value). Therefore, the larger the initial DNA template amount in the PCR reaction solution, the smaller the Ct value, and the smaller the template DNA amount, the larger the Ct value. Also, even if the amount of template DNA is the same, the greater the proportion of cleavage occurring in the specific gene of PCR within the template, the greater the Ct value of the PCR reaction in the same region.

The gene amplified from the test sample by the gene amplification method of the present invention may of course be subjected to detection or quantification. Alternatively, hybridization with beads, extension reaction using probe DNA, gene detection by hybridization, etc. can be mentioned, and detection using optical means may of course be used.

By using the method for amplifying genes and the method for detecting specific genes of the present invention, it is possible to detect single nucleotide polymorphisms (SNPs) on the 3 billion base pairs human genome gene sequence. In addition to examining the genetic background from type determination, it is also possible to diagnose the future risk of genetic diseases for which the causative gene is known. For example, genetic factors such as resistance to alcohol can be examined by SNP typing the alcohol dehydrogenase gene (ADH1B) and the aldehyde dehydrogenase gene (ALDH2).

(general technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art. : A Laboratory Manual, Cold Spring Harbor and its 3rd Ed.(2001); Ausubel, FM(1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM(1989). Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA(1990).PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM(1992).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al.(1995).PCR Strategies, Academic Press; Ausubel, FM(1999).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Supplementary Volume Experimental Medicine "Gene Introduction & Expression Analysis Experimental Method" Yodosha, 1997, etc., and these are the relevant parts of the present specification. (which may be all) are incorporated by reference.

DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes are described, for example, in Gait, M.J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M.J. (1990). Practical Approach, IRL Press; Eckstein, F.(1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R.L. et al.(1992). et al.(1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G.M. et al.(1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G.T.(I996). , which are incorporated herein by reference in relevant part.

In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "or". When we say "within a range" of "two values" in this specification, the range includes the two values themselves.

All references, such as scientific articles, patents, patent applications, etc., cited herein are hereby incorporated by reference in their entireties to the same extent as if each were specifically set forth.

The present invention has been described above with reference to preferred embodiments for ease of understanding. The present invention will now be described with reference to examples, which are provided for illustrative purposes only and not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but only by the claims.

Examples are described below. Where necessary, in the examples below, all experiments were performed in accordance with guidelines approved by the Mukogawa Women's University Ethics Committee. In addition, the ethical guidelines for human genome/gene analysis research prepared by the Ministry of Education, Culture, Sports, Science and Technology, the Ministry of Health, Labor and Welfare, and the Ministry of Economy, Trade and Industry (partially revised on November 25, 2014) were followed. In addition, it complied with the ethical guidelines for human genome gene analysis research. The experiment was carried out with approval after review by Mukogawa Women's University Ethics Committee. The reagents specifically used the products described in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical, Nacalai, R&D Systems, USCN Life Science INC, etc.) can be substituted.

(Example 1: Difference in amplification accuracy due to difference in sampling method)
(Sampling method)
Saliva (including human oral mucosal cells) was collected from test subjects (30 subjects) according to the following manual.
(1) A swab stick (made of polyurethane, manufactured by Texwipe, STX708A or STX712A) was held, and the outside and inside of the gums and the inside of the cheek were rubbed with one side of the sponge about 10 times. The other side was similarly rubbed about 10 times. Both sides of the swab were moistened with saliva.
(2) Holding the base of the stick, one side of the sponge was pressed against the center of a sampling plate (plastic plate with water-soluble paper attached) for 5 seconds, and the other side was similarly pressed for 5 seconds. Note: Do not scrape the plate (3) Return the used swab to its original bag and discard.
(4) The sampling sheet was folded in half and dried for 30 minutes or more. The sample plate was dried while being careful not to allow other people's saliva to adhere to the sample plate.

(TaqMan (registered trademark) PCR method)
In the TaqMan (registered trademark) PCR method, the alcohol dehydrogenase ADH1B gene and alcohol dehydrogenase ADH1B gene and Detection of genetic polymorphism in the gene of aldehyde dehydrogenase ALDH2 was performed by the TaqMan (registered trademark) probe method using a real-time PCR device ABI7300 (manufactured by Applied Biosystems). Saliva samples were collected using square swabs, round swabs, trust catchers or droppers, as described above, and transferred to water-dissolvable carriers as described herein for detection reactions. A sample was prepared for use.

In the TaqMan (registered trademark) probe method, an oligonucleotide called a TaqMan (registered trademark) probe is used in addition to a normal primer set for PCR amplification. TaqMan® probes are modified at one end with a fluorescent substance and at the other end with a quencher substance that suppresses fluorescence. As the DNA polymerase replicates in the PCR reaction, the TaqMan® probe specifically hybridized to the template DNA is degraded by the 5'-3' exonuclease activity of the replication enzyme. When the TaqMan® probe is degraded, fluorescence is emitted from the fluorescent molecule released from the quencher substance. In fluorescence detection, fluorescent molecules are excited by light from a tungsten halogen lamp to generate fluorescence, which passes through a filter that allows only the wavelength of the fluorescence to pass through and is detected by optical detection means such as a CCD camera.

Since the intensity of fluorescence indicates the amount of the probe hybridized to the template DNA, the change in the amount of template DNA can be tracked by detecting the intensity of fluorescence at each cycle of PCR. When the amount of the PCR amplification product reaches the fluorescence detectable amount, the amplification curve begins to rise, the signal rises exponentially, and then reaches a steady state. The greater the initial amount of template DNA, the faster the amount of amplified product reaches detectable amounts, and thus the faster the amplification curve rises. For example, the number of cycles to reach half the steady-state signal can be an indication of the initial amount of template DNA.

Reactions can also be performed with multiple TaqMan® probes specific for different sequences. Additionally, the fluorophores that modify each TaqMan® probe can produce different wavelengths of fluorescence, which can be differentially detected. Here, for alleles at a locus (for example, alleles X and Y), when a reaction is performed using TaqMan (registered trademark) probes specific for each, in one reaction, the present in the sample The amount of template DNA for X and Y can be determined, and the genotype (XX, YY, or XY) can be determined. For example, taking the fluorescence intensity derived from the allele X-specific probe on the X axis and the fluorescence intensity derived from the allele Y-specific probe on the Y axis, the fluorescence intensity in the amplification of the sample is calculated. By plotting, it is possible to determine the genotype from the position where the sample is plotted.

60MDP (manufactured by Nippon Paper Papyria Co., Ltd.) was used as a water-degradable carrier, which was impregnated with saliva (oral mucosal cells) as a test sample and allowed to dry naturally. 50 μL of sterilized water (DW: distilled water) was put into a tube-shaped insoluble carrier, and a test sample was supported on the dried water-decomposable carrier. After drying, a punch piece with a diameter of 4 mm was used (Biopsy trepan manufactured by Kaijirushi Co., Ltd. was used). was directly added and heated at 95° C. for 5 minutes (test sample preparation solution). 2 μL of the solution was added to another tubular insoluble carrier to prepare a reaction solution (each composition will be described later). Amplification reaction was carried out directly without the usual steps of DNA extraction and purification.

TaqMan (registered trademark) SNP Genotyping Assays kit from Applied Biosystems for probe and primer set, and THUNDERBIRD (registered trademark) Probe qPCR Mix (manufactured by Toyobo Co., Ltd.) for PCR amplification. was used to prepare 10 μL reaction solutions each containing the following reagents. 50X ROX reference dye (contained in THUNDERBIRD Probe qPCR Mix, Toyobo Co., Ltd.) was used as a reference for adjusting fluorescence. Color was determined by fluorescence intensity.
(TaqMan (registered trademark) probe/primer set for ADH1B gene)
- TaqMan (registered trademark) Drug Metabolism Genotyping Assays (manufactured by Applied Biosystems),
・Gene name: alcohol dehydrogenase 1B (class I), beta polypeptide
- TaqMan® SNP Genotyping Assay Mix ID: C_2688467_20.
(TaqMan® reaction solution for ADH1B gene)
THUNDERBIRD Probe qPCR Mix: 1 5 μL
Taqman® Assay Mix C_2688467_20: 0.5 μL
50X ROX reference dye: 0.2μL
DW (distilled water): 2.3 μL
Test sample preparation: 2 μL
Total volume: 10 μL
(TaqMan (registered trademark) probe/primer set for ALDH2 gene)
・ TaqMan (registered trademark) Drug Metabolism Genotyping Assays (manufactured by Applied Biosystems)
・Gene name: aldehyde dehydrogenase 2 family (mitochondrial)
- TaqMan® SNP Genotyping Assay Mix ID: C_11703892_10.
(TaqMan (registered trademark) reaction solution for ALDH2 gene)
THUNDERBIRD Probe qPCR Mix: 5 μL
Taqman® Assay Mix C_11703892_10: 0.5 μL
50X ROX reference dye: 0.2μL
DW (distilled water): 2.3 μL
Test sample preparation: 2 μL
Total volume: 10 μL
(amplification conditions)
Heat denaturation: 40 cycles of 50°C, 2 minutes → 95°C, 10 minutes, 95°C, 15 seconds → 60°C, 1 minute.

(result analysis)
Results were analyzed using ABI Prism 7300 SDS software.

(result)
The results are shown in Figures 1-2. FIG. 1 shows the results for ADH1B, and FIG. 2 shows the results for ALDH2. In the case of collecting saliva with a dropper, it was not possible to distinguish between them. Practically, the swab angle is convenient and advantageous.

Furthermore, blood and saliva samples were attached and dried on water-soluble paper (60 MDP), and blood was collected from Harris Micropunch (GE) with a diameter of 1.2 mm. Healthcare) and saliva samples were punched with a biopsy trepan (Kai) with a diameter of 4 mm, respectively, and subjected to real-time PCR for genotyping in the same manner. For 23 subjects, saliva was collected using a swab stick. Unambiguous genotyping was also possible in samples from these subjects (Figure 22).

(Example 2: Difference between water-soluble paper and qualitative filter paper)
In this example, experiments were conducted by comparing water-soluble paper and qualitative filter paper.

The experimental procedure was based on Example 1, and when the qualitative filter paper was used, the experiment was conducted by replacing the water-soluble paper with the qualitative filter paper. Saliva (approximately 20 μL) containing oral mucosa cells collected with a trust catcher was dropped, the dropping position was cut with a punch of 4 mm diameter (Φ), and heat treatment was performed. Other experimental procedures were the same as in Example 1.

As the water-soluble paper, 30MDP, 60MDP, 120MDP, 30CD-2, 60CD-2 and 120CD-2 (Nippon Paper Papyria) were investigated. As the filter paper, No. 1 filter paper manufactured by ADVANTEC was used. 1, No. 2, No. 3, No. 4A and no. 5 of five standards were examined.

The results are shown in FIG. It was found that the amount of cells retained was almost constant regardless of the paper quality of the water-soluble paper. From the viewpoint of operability, 60 MDP was selected for the experiment. On the other hand, in the filter paper, No. 4, which has a low water absorption 4A was found to have a low cell retention amount.

Furthermore, the SYBR® Green real-time assay method compared the performance of a functional water-based paper (60MDP, Nippon Paper Papilia) to a qualitative filter paper (ADVANTEC No. 1) and Whatman® 903 protein saver card. FIG. 21 shows the evaluation results. It is observed that the Ct value (25 cycles) for water paper is amplified 5 cycles earlier than for qualitative filter paper and protein saver cards. As a negative control, amplification was performed by adding distilled water (DW). Primer dimers and non-specific gene amplification are observed after 35 cycles.

(Example 2A: Various carriers)
In this example, in addition to water-soluble paper and qualitative filter paper, experiments were conducted using other carriers. The experimental procedure was based on Example 1, and experiments were conducted by replacing the water-soluble paper with Kimwipe (Nippon Paper Crecia), Kimtowel (Nippon Paper Crecia), newspaper (Yomiuri Shimbun) and tissue paper (Kleenex, Nippon Paper Crecia).

The results are shown in Figure 3A. These results demonstrate that not only water-soluble paper or qualitative filter paper, but also general paper can be used as a carrier in the present invention as long as it exhibits water absorbency.

(Example 2B: Difference in base material/sampling method)
In this example, differences when using various base materials/sampling methods, and differences due to the presence or absence of heating after putting the carrier into sterilized water were verified.

When a Kimwipe was used as the base material, the Kimwipe was held in the mouth, rolled in the oral cavity, rolled, sufficiently moistened with saliva, stamped, and dried.

When a swab is used as a base material, (i): the swab (0) is obtained by holding the swab in the oral cavity and absorbing saliva, and (ii): collecting saliva by rubbing the mucous membrane in the oral cavity once with the swab. Swab (1) was swab (1), and (iii): swab (2), swab (4), and swab (∞) were obtained by changing the conditions of (ii) for 2 and 4 laps, respectively. and

Cells were transferred to the carrier by pressing one side of these substrates against water-soluble paper for 5 seconds. Other experimental procedures were based on Example 1, and 50 μL of sterilized water (DW: distilled water) was added to the tube-shaped insoluble carrier, and the test sample was supported on this. (using Kaijirushi biopsy trepan) was directly added and heated at 95°C for 5 minutes, and the results were compared with those without heating.

The results are shown in Figures 3B-3G. When a swab was used as the substrate, the genotype discrimination ability was slightly improved by heating (Fig. 3G). Furthermore, when newspaper was used as the base material, the genotype discrimination ability was significantly improved when heated compared to when it was not heated (Fig. 3B).
The swabs showed high genotype discrimination, independent of the number of times the oral cavity was rubbed (FIGS. 3D and 3E). Therefore, this result suggests that the surface area of the sampling surface, rather than the volume to be sampled, is important for sampling.

In addition, detailed differences were investigated for swab-like substrates. Using a swab (Texwipe, STX712A), a swab short form type (AS ONE HJ-3329), a cotton swab, PVA (a saliva collection part of a trust catcher was used in the same manner as the swab and transferred to a carrier) as a base material, oral mucosa Cells were harvested and transferred by pressing onto water-soluble paper. In addition, the genotype was determined in the same procedure as above.

The results are shown in Figure 3F. Sufficient signals have been detected using any substrate, and any of these substrates can be used in the present invention.

(Example 2C: Suspension and supernatant)
In this example, the difference between the case where a swab was used as a base material, the carrier was placed in sterilized water and then subjected to the PCR reaction while suspended, and the case where only the centrifuged supernatant was subjected to the PCR reaction. verified. The experimental procedure was based on Example 1, but after putting the carrier into sterilized water, the suspension was centrifuged (15,000×rpm) and only the supernatant was subjected to PCR reaction.

The results are shown in Figure 3G. No difference in genotype discrimination was observed between the supernatant and the suspension. Therefore, this result indicates that the kit of the present invention enables the preparation of a sample capable of sufficiently identifying the genotype in a state in which the carrier carrying the test sample is suspended.

(Example 2D: Comparison of swab (polyurethane) and applicator (trust catcher))
Using a swab (polyurethane, Texwipe, STX708A or STX712A) as a substrate and an applicator (polyvinyl alcohol trust catcher (registered trademark)), ADH1B polymorphisms of 6 subjects were analyzed.

Sampling was performed by rubbing the inside of the oral cavity with a swab and applicator and then pressing it against water-soluble paper. Subsequently, the ADH1B gene polymorphism was analyzed using the TaqMan (registered trademark) PCR method in the same manner as in the above Examples. The applicator is a member that absorbs saliva due to its structure, and has an absorbent part attached to its tip. The inner cheek was rubbed with the tip of the swab in the same manner as a swab, and pressed vertically against water-soluble paper to adhere saliva containing oral mucosal cells in PVA.
(Composition of reaction solution of swab sample)
20×Probe (ADH1B) 0.5μL
50×ROX reference dye 0.2μL
THUNDERBIRD Probe qPCR Mix: 5 μL
Φ4mm in DW (distilled water) 4.3μL
Total 10.0μL
(*) A sample punched with a diameter of 4 mm was placed in 100 μL of distilled water and heated at 95° C. for 5 minutes with THERMO BLOCK ND-G962 (manufactured by Nisshin Rika). Storage was at -20°C.
(Composition of reaction solution of applicator sample)
20×Probe (ADH1B) 0.5μL
50×ROX reference dye 0.2μL
THUNDERBIRD Probe qPCR Mix: 5 μL
1 μL of KODFX NEO
Φ6mm in DW (distilled water) 4.3μL
Total 10.0μL
(*) A sample punched with Φ6 mm was placed in 200 μL of distilled water, and the sample to be heated was heated at 95° C. for 5 minutes with THERMO BLOCK ND-G962 (manufactured by Nisshin Rika).
(Amplification conditions)
The real-time PCR cycle was as follows.
Program:7300, linda, ADH1B_TaqMan (registered trademark)_kit
50°C, 2 minutes → 95°C, 10 minutes → (95°C, 15 seconds → 60°C, 1 minute) x 40 cycles (result)
The results are shown in Figure 3H. Diamonds indicate samples rated A, triangles indicate samples rated A/G, and circles indicate A/G positive controls (pc). The swab and applicator determined 6 subjects as black diamonds ◆ (A) (2 people) and black triangles ▲ (A/G) (4 people), indicating that they are clearly separated. Black diamonds ◆ indicate equally good sensitivity between swab and applicator. Therefore, both polyurethane and PVA can be used as substrate materials for genotyping. And it can be interpreted that even an applicator that is not originally intended to be used in this way can be similarly used. It was also suggested that preferred embodiments should have a swab-like shape in which the surface area does not fluctuate, rather than an applicator-like shape in which the surface area fluctuates upon contact or application.

The combination of polyurethane swab and water-soluble paper showed very high sensitivity in all the samples of the black triangle subjects, and can be said to be very good. In the case of the applicator, slightly less sensitivity is observed in some of the black triangles, which, without being bound by any theory, is impregnated with saliva in a constant volume of substrate. However, this may be due to the presence of slight variations due to changes in the contact area caused by the strength of pressing when transferring to the water-soluble paper. When attempting to perform quantitative sampling in a constant volume using a thick-shaped base material, variations in quantitative performance can occur in this way. The swab has a flat shape with little thickness, and when it comes into contact with the water-soluble paper, the contact area is constant regardless of the pressing force, etc., which may have led to better quantification. .

(Example 3: Real-time PCR melting curve analysis method)
In this example, it was confirmed whether the present invention can be applied to real-time PCR melting curve analysis. The procedure etc. are shown below. In this example, an experiment was conducted using a melting curve (dissociation curve). Briefly:

(sample)
Saliva (containing human oral mucosal cells) was collected from test subjects (30 subjects) in the same manner as in Example 1 according to the following manual.
(1) Hold a swab stick (same as in Example 1) and rub the outside and inside of the gums and the inside of the cheek with one side of the sponge about 10 times. The other side was similarly rubbed about 10 times. Both sides of the swab were moistened with saliva.
(2) Holding the base of the stick, one side of the sponge was pressed against the center of the sampling plate (plastic plate with water-soluble paper attached) for 5 seconds, and the other side was similarly pressed for 5 seconds. do not rub).
(3) The used swab was returned to the original bag and discarded. (4) The sampling sheet was folded back in half and dried for 30 minutes or longer. Carefully dry the sample plate so that other people's saliva does not stick to the sample plate.
(Method)
In real-time PCR by the intercalator method, a method of confirming the Tm value of PCR amplification products was used. In the melting curve analysis, after the PCR reaction, the temperature of the reaction solution is gradually increased from 60°C to 95°C and the fluorescence value is monitored. Strong fluorescence is detected when the PCR product forms double strands, but when it reaches a certain temperature (Tm value), it dissociates into single strands and the fluorescence value drops sharply. Since the Tm value varies depending on the length and GC content of the PCR product, it is possible to distinguish between the target amplification product and short amplification products such as primer dimers.

Angiotensin converting enzyme (ACE)
Angiotensin II is converted from angiotensin I by angiotensin converting enzyme (ACE). The ACE gene, which encodes ACE, is located on the long arm of chromosome 17 (17q23) in humans and contains 26 exons and 25 introns. The ACE gene has an insertion allele (I) containing a 287bp DNA fragment in the 16th intron and a deletion allele (D) lacking this, and is classified into three genotypes, II, ID and DD. In 1990, Rigat et al. reported that each genotype had different serum ACE levels, with D allele having higher levels than I allele. (J Clin Invest 1990;86:1343-1346)
(References) Nakahara K, Matsushita S., Matsuoka H., Inamatsu T., Nishinaga M., Yonawa M., Aono T., Arai T., Ezaki Y. and Orimo H. Insertion/depletion polymorphism in the angiotensin converting Enzyme gene affects heart weight. Circulation 2000; 101(2): 148-151
For PCR amplification, KOD (registered trademark) SYBR (registered trademark) qPCR Mix (manufactured by Toyobo Co., Ltd.) was used to prepare 20 μL reaction solutions each containing the following reagents. Genotyping was determined by melting curve analysis of the results of this reaction.
(reagent)
KOD SYBR® qPCR Mix: 10 μL
Forward Primer 1.0 μL (10 pmol)
Reverse Primer 1.0 μL (10 pmol)
50X ROX reference dye: 0.4μL
DW (distilled water): 2.6μL
Test sample preparation: 5 μL
Total volume: 20 μL
(Primer)
Forward Primer: 5'-CTGGAGACCACTCCCATCCTTTCT-3' (SEQ ID NO: 1)
Reverse Primer: 5'-ATGTGGCCATCACATTCGTCGTCAGAT-3' (SEQ ID NO: 2)
(amplification conditions)
Thermal denaturation: 40 cycles of 98°C for 2 minutes → 98°C for 10 seconds → 60°C for 10 seconds → 68°C for 1 minute.

(melting curve)
95°C 15 seconds → 60°C 1 minute → 99°C 15 seconds → 60°C 15 seconds (result)
The results are shown in FIG. As shown in the figure, the results were Insert (490 bp): I/I 87.2°C, Delete (190 bp): D/D 80.7°C, I/D 80.7°C and 87.2°C. This result indicated that genotype differences can be easily detected. From this example, it can be said that the present invention improved the simplicity and sensitivity. That is, even without DNA extraction, since the DNA is amplified in 20 to 30 cycles, highly sensitive gene determination is possible, and it is understood that the present invention enables simple quantitative analysis.

Furthermore, for the test subjects (16 subjects), using a different instrument (QuantStudio ^™ 12K Flex Real-Time PCR System (Thermo Fisher Scientific Inc.)), dry saliva samples using water-based paper (120 MDP) were used to determine the ACE genotype. When determination was performed, clear genotype determination was possible by melting curve (Fig. 23). From this, it is understood that the sampling substrate of the present invention is compatible with real-time PCR using various instruments.

(Example 3A: Detection of PCR products by electrophoresis)
Furthermore, it was confirmed that the ACE genotype can be successfully determined by subjecting the PCR amplified product to electrophoresis using the sample prepared according to the present invention. The results of analyzing the real-time PCR product of Example 3 using a microchip electrophoresis device MultiNA (Shimadzu) are shown in FIG. matches the result of

Since the I allele is amplified as a 490 bp fragment and the D allele as a 190 bp fragment using the primers of SEQ ID NOs: 1 and 2, 490 bp (II), 190 bp (DD), 190 bp and 490 bp (ID) are detected. can. The ACE genotype can be determined by subjecting the PCR amplification products to electrophoresis and size separation (for example, using a microchip electrophoresis device MultiNA (Shimadzu)) (Fig. 4A). Methods of using such primers are also described, for example, in the following references.

High frequency of the Dallele of the angiotensin-converting enzyme gene in Arabic populations BMC Research Notes 2009, 2:99
Angiotensin-converting enzyme insertion/deletion gene polymorphisms is associated with risk of glioma in a Chinese population Journal of the Renin-Angiotensin-Aldosterone System 0(2016)
Endothelial nitric oxide synthase, angiotensin-converting enzyme and angiotensinogen gene polymorphisms in hypertensive disorders of pregnancy Hypertension Research (2010) 33, 473-477
Insertion-Deletion Polymorphism of the ACE Gene Modulates Reversibility of Endothelial Dysfunction With ACE Inhibition Circulation July 4, 2000
ACE I/D and ACTN3 R/X polymorphisms as potential factors in modulating exercise-related phenotypes in older women in response to a muscle power training stimuli Age(Dordr) 35(5) (2013) 1949-1959
Angiotensin-converting enzyme and angiotensin II receptor 1 polymorphisms: association with early coronary disease Cardiovascular Research 40 (1998) 375-379
Association Between the Polymorphism of the Angiotensin - Converting Enzyme Gene and Breast Cancer Risk Among the Bengalee Caste Hindu Females of West Bengal, India Int J Forensic Sci Pathol. 3(2), 85-88.
The Angiotensin I-Converting Enzyme Gene Insertion/Deletion Polymorphism Is Linked to Early Gastric Cancer Cancer Epidemiol Biomarkers Prev 2005;14(12).
ROLE OF ANGIOTENSIN-CONVERTING ENZYME INSERTION/DELETION POLYMORPHISM IN TYPE I DIABETES NEPHROPATHY Romanian Journal of Diabetes Nutrition & Metabolic Diseases / Vol. 19 / no. 2 / 2012
Angiotensin-converting enzyme gene insertion/deletion polymorphism in migraine patients BMC Neurology 2008, 8:4.

(Example 4: Copy number variation test by real-time PCR)
In this example, copy number variation (CNV) was tested by real-time PCR.

(sample)
Saliva (containing human oral mucosal cells) was collected from test subjects (30 subjects) in the same manner as in Example 1 according to the following manual.
(1) Holding a swab stick, one side of the sponge rubbed the outside and inside of the gums and the inside of the cheek about 10 times. The other side was similarly rubbed about 10 times. Both sides of the swab were moistened with saliva. (2) Holding the base of the stick, one side of the sponge was pressed against the center of the sampling plate (plastic plate with water-soluble paper attached) for 5 seconds, and the other side was similarly pressed for 5 seconds. do not rub).
(3) Used swabs were returned to their original bags and discarded.
(4) The sampling sheet was folded in half and dried for 30 minutes or longer. The sample plate was dried while being careful not to allow other people's saliva to adhere to the sample plate.

(Method)
In recent years, copy number variations (CNVs) caused by genomic structural changes have been pointed out to be associated with diseases and drug responsiveness. In response to this, methods (DNA chips, sequencers, etc.) to search for CNVs on a genome-wide basis have been developed, and databases have been constructed as a result of the efforts of researchers around the world, and the information is updated daily. On the other hand, however, there are not so many technologies that allow anyone to quantitatively validate target CNVs in many specimens. TaqMan® Copy Number Assays are the preferred method for validating CNVs, with over 1.6 million TaqMan® probes and primers designed for humans and TaqMan ( Copy Number Assays (Registered Trademark) and TaqMan (Registered Trademark) Copy Number Reference Assays serving as an internal control can be used to determine copy number in 3-4 hours on a real-time PCR instrument.

In recent years, large-scale deletions, duplications, and insertions of nucleotide sequences of more than 1,000 bases on the genome have occurred over a wide range. In order to elucidate individual differences in diseases from a pharmacogenomic approach, it is necessary to collect not only SNPs but also CNV information from various fields. However, the expression frequencies of these SNPs and CNVs differ among races and ethnic groups, suggesting the importance of collecting race- and ethnic-specific SNP and CNV information. Therefore, it is necessary to build a foundation for personalized medicine based on genomic information in Japan by accumulating, scrutinizing, and evaluating the expression frequencies of SNPs and CNVs of various drug susceptibility genes in Japanese.

In general, humans inherit two sets of genes, one from each parent's genome. Normally, when focusing on a certain gene, it has been thought that each gene has two genes. However, in recent years, it has become clear that there are individual differences in the number of gene copies (copy number polymorphism), such as the presence of only one copy of a gene or three or more copies of a gene per cell, depending on the individual. This copy number polymorphism is attracting attention as a cause of individual differences in constitution, such as differences in efficacy and side effects of various drugs.

CNVs were determined by the TaqMan (registered trademark) Copy Number Assay method as follows. The assay ID of the TaqMan (registered trademark) Copy Number Assay (Life Technologies, Inc.) used was Hs00010001_cn for CYP2D6, and RNase P was used for the TaqMan (registered trademark) Copy Number Reference Assay. The ABI PRISM (registered trademark) 7300 Fast Real-Time PCR System was used to amplify template DNA from dried saliva in the same manner as TaqMan (registered trademark) SNP typing, and the copy number was determined from the ΔΔCt of the amplification curve.

The composition of the PCR reaction solution is as follows: test sample preparation solution: 2 μL, THUNDERBIRD (registered trademark) Probe qPCR Mix: 5 μL, 20 × TaqMan (registered trademark) Copy Number Assay 0.5 μL, 20 × TaqMan (registered trademark) Copy Number Reference Assay 0.5 μL, 50X ROX reference dye: 0.2 μL, sterile purified water added to a total volume of 10 μL (see below).
THUNDERBIRD Probe qPCR Mix: 5 μL
20x CNV Probe (CYP2D6*5, Hs00010001_cn) : 0.5μL
20x Copy Number Reference Assay RNaseP: 0.5μL
50X ROX reference dye: 0.2μL
DW (distilled water): 1.8 μL
Test sample preparation: 2 μL
Total volume: 10 μL
(amplification conditions)
Heat denaturation: 40 cycles of 50°C, 2 minutes → 95°C, 10 minutes, 95°C, 15 seconds → 60°C, 1 minute.
(result)
FIG. 5 shows the results of copy number variation analysis of CYP2D6 by TaqMan (registered trademark) PCR. As shown, the amplification curve can be clearly distinguished between the case of 1 copy and the case of 2 copies, so it is possible to determine how many copies exist from the state of the curve. Here, it was shown that it can be used as a method for detecting copy number variation of CYP2D6. The defect was expressed as CYP2D6*5.

(Example 5: Example of sample collection from a remote location using a sampling kit)
In this example, a sampling kit was used to construct a remote sample collection method.

(Preparation for sampling (see Fig. 14))
As shown in the left column of FIG. 14, a sampling sheet and a swab (enclosed in a sterilized bag) are prepared and sent to the subject. The sheet is attached with double-sided tape that can be peeled off any number of times, and is fixed on water-soluble paper with double-sided tape.

Subject removes swab from plastic bag and opens sampling sheet.

Here, the subject is shown the following precautions.

(Notes)
Foreign objects in the mouth (e.g. food and drink) → Gargle.
(2) Immediately after drinking coffee → Gargle.
(3) Do not attach lipstick or gloss to the water-soluble paper.

Sampling method (see Figure 14)
(1) Hold a swab stick and rub the outside and inside of the gums and the inside of the cheeks with one side of the sponge about 10 times. Repeat on the other side about 10 times. Make sure both sides of the sponge are sufficiently wet.
(2) Hold the base of the stick, apply one side of the sponge to the ○ mark on the water-soluble paper, and press for 5 seconds. Do the same for the other side for 5 seconds. (Caution) Do not rub water-soluble paper.
(3) Return the swab to its original bag and dispose of it as garbage.
(4) Dry the water-soluble paper for at least 30 minutes. Be careful not to let dust or other people's saliva adhere to the water-soluble paper, and let it dry. Fold in half after drying.

(Consent form creation)
Next, the subject is asked to prepare a consent form to agree to the informed consent for this study. The consent form may be prepared as an instructional saddle-stitched consent form with a title such as "Alcoholism Check" (for ADH1B and ALDH2).
(Sample mailing)
(1) The subject peels off one sample ID sticker and pastes it on the back of the text.
(2) The subject puts the sampling sheet and consent form in an envelope and mails it. (eg, one that can encapsulate that of FIG. 14).

(Example 6: Confirmation of performance of oral mucosal cell-collecting carrier)
Newspaper, qualitative filter paper no. 1, and water-soluble paper 60 MDP punched with a diameter (Φ) of 6 mm, 5 μL of saliva was dropped, and the sample dried for 1 hour was placed in 200 μL of distilled water, every 1, 2, 5, 10, 30, 60 minutes. 5 μL of the supernatant removed at each time point after agitation and 5 μL of the supernatant removed after 60 minutes of incubation at 95° C. for 5 minutes were added to the TaqMan® PCR reaction solution and subjected to real-time PCR. 50X ROX reference dye was used as a reference for adjusting fluorescence. Thunderbird (Toyobo) (QPS-201X5 for 1000 times (5 sets of QPS-201)) was purchased and used.

The composition of the reactants was as follows.
20×Probe (ADH1B and ALDH2) 0.5 μL
50×ROX reference dye 0.2μL
THUNDERBIRD Probe qPCR Mix: 5 μL
Φ4mm in DW (distilled water) 4.3μL
Total 10.0μL
(*) A sample punched with Φ6 mm was placed in 200 μL of distilled water, and the sample to be heated was heated at 95° C. for 5 minutes with THERMO BLOCK ND-G962 (manufactured by Nisshin Rika).

The real-time PCR cycle was as follows.
50°C, 2 minutes→95°C, 10 minutes→(95°C, 15 seconds→60°C, 1 minute)×40 cycles Therefore, the copy number was theoretically calculated and used as a reference. For ADH1B, dilutions (10-fold serial dilutions) of approximately 5 million copies/μL of purified plasmid DNA were used as controls. In general, quantitative PCR reactions quantify in the range of 10 copies to 1 million copies. Quantification is best when the Ct value is between 20 and 30 cycles, which is in the range of 1,000 to 100,000 copies in terms of copy number. A 10-fold dilution series of a plasmid containing the target gene was subjected to a PCR reaction and used as a positive control.

FIG. 15 shows the results for newspaper. FIG. 16 shows the results of qualitative filter paper. FIG. 17 shows the results for water soluble paper. By heat treatment, all of newspapers, qualitative filter papers, and water-soluble papers release the collected sample, and a signal sufficient for genotype detection is obtained. Moreover, the signal intensity does not depend on the time during which the carrier is immersed in distilled water or the degree of stirring.

Water-soluble paper is particularly desirable because sufficient signals can be obtained without heat treatment. In particular, it has been shown to have good quantitation as it releases all or most of the bound sample.

(Example 7: Confirmation of no effect on quantification of number of transfers from swab to water-soluble paper)
Real-time PCR analysis was performed in the same manner as in Example 6 using a sample obtained by pressing one side of the same swab from which the oral mucosal cells were collected by successively pressing against four sheets of water-soluble paper for 5 seconds and drying. The water-soluble paper was cut into 4 mmΦ, placed in 100 μL of distilled water, and then heat-treated at 95° C. for 5 minutes. The reaction composition for real-time PCR was as described in Example 6 above.

Results are shown in FIG. It can be seen that most of the sample collected on the swab is transferred to the water-soluble paper with a single contact. That is, the transfer efficiency from the swab to the water-soluble paper does not depend on the number of times, and can be quantitatively measured.

(Example 8: Saliva concentration gradient experiment)
10 μL of saliva diluted stepwise in a 10-fold dilution series is dropped onto 60 MDP water-soluble paper cut to 6 mmΦ, dried for 1 hour, added to distilled water, and heat-treated at 95 ° C. for 5 minutes to remove the supernatant. 5 μL was subjected to a real-time PCR reaction similar to that described in Example 6. In addition, a sample obtained by adding 10 μL of the diluted saliva solution to distilled water and performing heat treatment in the same manner was similarly subjected to the real-time PCR reaction.

Results are shown in FIG. With water-soluble paper, the signal of the dilution series is linear and quantitative. Comparing saliva dripped and dried on water-soluble paper with saliva directly heat-treated, water-soluble paper has about 10 times better sensitivity. Therefore, it was also confirmed in this experiment that quantification is ensured if the surface area is constant. Furthermore, the quantification will be further detailed with reference to FIGS. 19A and 19B.

FIG. 19A shows a calibration curve based on the amplification curve of the positive control in the quantitative test of the sample released from water-soluble paper into water. The vertical axis is the Ct value and the horizontal axis is the copy number. The copy number of the positive control can be spectroscopically calculated from the purified DNA (copy number per μL). Linearity is shown for both alleles 1 and 2. The straight line shown in Figure 19A gave a regression rate of about 0.95 or greater.

FIG. 19B is a copy calculated using the calibration curve shown in FIG. 19A in a quantitative test of a sample released into water from water-soluble paper using saliva serially diluted in a 10-fold dilution series. It is a table showing numbers. When dropped onto water-soluble paper, linearity was exhibited up to 1000-fold dilution.

Therefore, it was also confirmed in this experiment that if the surface area is constant, quantification is ensured even at a considerable dilution ratio of at least 1000 times.

(Example 9: Comparison of water-soluble paper (60 MDP and 120 MDP))
Gene amplification was confirmed for ADH1B by a protocol similar to that described in Example 6, using water-soluble paper (60 MDP and 120 MDP) as carriers. Dried saliva samples were prepared using swabs as described in Example 5. In the present example, 10 μL of blood was dropped on water-soluble paper, dried, and then punched out to 5 mm.

As shown in FIG. 24, when saliva was used, gene amplification was confirmed in both water-soluble papers, and it is understood that any water-soluble paper can be used as a carrier regardless of the type of water-soluble paper. In the case of blood samples, it was confirmed that gene amplification was greater when 120 MDP was used (FIG. 25). This indicates that thicker water-soluble paper may be advantageous for samples such as blood, which may contain many amplification inhibitors. In addition, the 120 MDP water-soluble paper has sufficient strength without being fixed on the backing paper when creating the sampling card, and it was possible to make cuts in advance, so it was possible to cut without using a punch or the like. It was possible to cut out a constant area by extrusion (Fig. 14).

(Note)
While the invention has been illustrated using the preferred embodiments thereof, it is understood that the invention is to be construed in scope only by the appended claims. It is understood that the patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety to the same extent as if the content itself were specifically set forth herein. understood. This application claims priority to Japanese Patent Application No. 2016-152125 filed on August 2, 2016 with the Japan Patent Office. Incorporated as

INDUSTRIAL APPLICABILITY The present invention can be used as a drug and its development, for example, as a simple diagnostic agent, and can be utilized as a simple companion diagnostic agent in the future.

SEQ ID NO: 1: Forward Primer used in Example 3
SEQ ID NO: 2: Reverse Primer used in Example 3

Claims (35)

A kit for preparing a sample for detecting copy number variation by amplifying nucleic acids from a sample containing a nucleic acid-containing biological material,
(A) a carrier capable of binding a nucleic acid-containing biomaterial;
(B) a substrate having lower binding ability for the nucleic acid-containing biomaterial than the carrier, wherein the carrier is water-soluble paper, the substrate is a swab, and the substrate is a nucleic acid-containing biomaterial in a certain amount. wherein the carrier and the substrate are configured to contact such that the contact area is constant, and the substrate has a varying surface area upon contact with the carrier No, Kit. 2. The kit of claim 1 , wherein the surface area of said substrate (B) is sufficient to provide said range of amounts. 3. The kit according to claim 1 or 2 , wherein said carrier (A) allows nucleic acid amplification when placed in water. The kit according to any one of claims 1 to 3 , wherein said carrier (A) allows optical detection. The kit according to any one of claims 1 to 4 , wherein nucleic acids in said sample after said nucleic acid amplification are optically detected. The kit according to any one of claims 1 to 5 , wherein said carrier (A) is a water-decomposable carrier. The kit according to any one of claims 1 to 6 , wherein the fixed range amount is an amount capable of discriminating the copy number after nucleic acid amplification. Claims 1 to 1, wherein the amount within a certain range is within X times to 1/X of the reference amount, and ^X2 is a identifiable fold difference when the copy number differs by 1 in the nucleic acid amplification . 8. The kit according to any one of 7 . The kit according to any one of claims 1 to 8 , wherein the substrate (B) is a substrate that supports a nucleic acid-containing biomaterial without binding. The base materials include polyurethane, polyvinyl alcohol (PVA), polyethylene, chitin, chitosan, collagen, polyvinylidene difluoride (PVDF), polypropylene, cellulose and its derivatives, paper, collagen, gelatin, urethane, silicon and absorbent cotton, or combinations thereof , or mixtures thereof. The kit according to any one of claims 1 to 10 , wherein the range of amounts is achieved by taking out a constant area of the carrier. 12. The kit according to claim 11 , wherein the surface area of said substrate (B) is equal to or greater than said fixed area. 13. A kit according to claim 11 or 12 , wherein said substrate (B) has a shape sufficient to cover said fixed area. The kit according to any one of claims 11 to 13 , wherein said constant area is achieved by cutting said constant area with a cutting device capable of cutting said constant area. 15. The kit of Claim 14 , wherein said resector is selected from a biopsy trepan, and a punch. The kit of any one of claims 1-15 , wherein said range of amounts is achieved by providing said sample in a constant amount. The kit according to any one of claims 1 to 16 , wherein said carrier is water-disintegrable paper. The kit according to any one of claims 1 to 17 , wherein said nucleic acid amplification is a nucleic acid amplification method in which non-specific amplification occurs. A kit for amplifying nucleic acids without purification, comprising the kit according to any one of claims 1 to 18 and (C) reagents for nucleic acid amplification. 20. The kit of claim 19 , wherein the reagents for nucleic acid amplification comprise primers, nucleic acid amplification enzymes, and necessary buffers. The kit according to any one of claims 1 to 20 , wherein said (A) further comprises a non-water absorbent material and a water absorbent material. 22. The kit of claim 21 , wherein said non-water absorbent material is arranged to hold said carrier, said non-water absorbent material being removably coupled to said water absorbent material. 23. The kit according to claim 21 or 22 , wherein said non-water absorbent material is connected to said water absorbent material via a removable adhesive member. A kit according to any one of claims 21 to 23 , wherein said non-water absorbent material is arranged to surround said carrier. The kit of any one of claims 21-24 , wherein the carrier contacts the non-bibulous material and the bibulous material. A kit according to any one of claims 21 to 25 , wherein said carrier is arranged so as not to be in direct contact with said water-absorbing material. A method for detecting the copy number of a specific gene contained in a nucleic acid-containing biomaterial, comprising:
A step of providing a test sample containing the nucleic acid-containing biomaterial by using (A) a carrier capable of binding the nucleic acid-containing biomaterial and (B) a substrate having a lower binding ability for the nucleic acid-containing biomaterial than the carrier. ,
a step of contacting the carrier with an aqueous solution containing a nucleic acid amplification reagent containing a primer that enables specific amplification of the specific gene, and subjecting the carrier to conditions for nucleic acid amplification;
detecting the copy number of the specific gene using the optical means , wherein the carrier is water-soluble paper, the substrate is a swab, and the substrate is a nucleic acid-containing biomaterial in a certain amount. wherein the carrier and the substrate are configured to contact such that the contact area is constant, and the substrate has a varying surface area upon contact with the carrier no way. 28. The method of claim 27 , wherein a test sample comprising the nucleic acid-containing biomaterial is provided by contacting the substrate with the nucleic acid-containing biomaterial and then contacting the substrate with the carrier. 29. The method of claim 27 or 28 , wherein the test sample is provided to contain a range of amounts of nucleic acid-containing biomaterial. A kit for providing a sample for detecting the copy number of a specific gene contained in a nucleic acid-containing biomaterial using optical means,
For providing a test sample containing a nucleic acid-containing biomaterial, comprising (A) a carrier capable of binding a nucleic acid-containing biomaterial, and (B) a substrate having a lower affinity for the nucleic acid-containing biomaterial than the carrier. a kit, primers that enable specific amplification of the specific gene,
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
wherein the carrier is water -based paper, the substrate is a swab, the substrate is configured to provide a sample containing a range of amounts of nucleic acid-containing biological material, and the carrier and the substrate are A kit configured for constant contact area contact, wherein said substrate does not vary in surface area upon contact with said carrier. A system for providing a sample for detecting the copy number of a specific gene contained in a nucleic acid-containing biomaterial using optical means,
For providing a test sample containing a nucleic acid-containing biomaterial, comprising (A) a carrier capable of binding a nucleic acid-containing biomaterial, and (B) a substrate having a lower affinity for the nucleic acid-containing biomaterial than the carrier. a kit and primers that enable specific amplification of the specific gene;
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
a nucleic acid amplification device, wherein the carrier is water-soluble paper, the substrate is a swab, the substrate is configured to provide a sample containing a range of amounts of nucleic acid-containing biological material, the carrier and the A system configured to contact a substrate with a constant contact area, wherein the substrate does not vary in surface area upon contact with the carrier. A system for detecting the copy number of a specific gene contained in a nucleic acid-containing biomaterial,
For providing a test sample containing a nucleic acid-containing biomaterial, comprising (A) a carrier capable of binding a nucleic acid-containing biomaterial, and (B) a substrate having a lower affinity for the nucleic acid-containing biomaterial than the carrier. a kit, primers that enable specific amplification of the specific gene,
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
a nucleic acid amplification device and optical means, wherein the carrier is water-soluble paper, the substrate is a swab, the substrate is configured to provide a sample containing a range of amounts of nucleic acid-containing biological material; A system wherein the carrier and the substrate are configured to contact with a constant contact area, and wherein the substrate does not vary in surface area upon contact with the carrier. A kit for providing a sample for detecting the copy number of a specific gene contained in a nucleic acid-containing biomaterial,
A substrate containing a nucleic acid-containing biomaterial, comprising a device comprising a non-water-absorbing material, a carrier capable of binding a nucleic acid-containing biomaterial, and a water-absorbent resin, and a substrate having a lower binding ability for the nucleic acid-containing biomaterial than the carrier a kit for providing a test sample and primers that allow specific amplification of the specific gene;
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
wherein the carrier is water -based paper, the substrate is a swab, the substrate is configured to provide a sample containing a range of amounts of nucleic acid-containing biological material, and the carrier and the substrate are A kit configured for constant contact area contact, wherein said substrate does not vary in surface area upon contact with said carrier. A system for providing a sample for detecting the copy number of a specific gene contained in a nucleic acid-containing biomaterial,
A substrate containing a nucleic acid-containing biomaterial, comprising a device comprising a non-water-absorbing material, a carrier capable of binding a nucleic acid-containing biomaterial, and a water-absorbent resin, and a substrate having a lower binding ability for the nucleic acid-containing biomaterial than the carrier a kit for providing a test sample and primers that allow specific amplification of the specific gene;
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
a nucleic acid amplification device, wherein the carrier is water-soluble paper, the substrate is a swab, the substrate is configured to provide a sample containing a range of amounts of nucleic acid-containing biological material, the carrier and the A system configured to contact a substrate with a constant contact area, wherein the substrate does not vary in surface area upon contact with the carrier. A system for detecting the copy number of a specific gene contained in a nucleic acid-containing biomaterial,
A substrate containing a nucleic acid-containing biomaterial, comprising a device comprising a non-water-absorbing material, a carrier capable of binding a nucleic acid-containing biomaterial, and a water-absorbent resin, and a substrate having a lower binding ability for the nucleic acid-containing biomaterial than the carrier a kit for providing a test sample and primers that allow specific amplification of the specific gene;
a nucleic acid amplification reagent or an aqueous solution containing a nucleic acid amplification reagent;
a nucleic acid amplification device and optical means, wherein the carrier is water-soluble paper, the substrate is a swab, the substrate is configured to provide a sample containing a range of amounts of nucleic acid-containing biological material; A system wherein the carrier and the substrate are configured to contact with a constant contact area, and wherein the substrate does not vary in surface area upon contact with the carrier.